Multiple electrospray device, systems and methods

ABSTRACT

A microchip-based electrospray device, system, and method of fabrication thereof are disclosed. The electrospray device includes a substrate defining a channel between an entrance orifice on an injection surface and an exit orifice on an ejection surface, a nozzle defined by a portion recessed from the ejection surface surrounding the exit orifice, and an electric field generating source for application of an electric potential to the substrate to optimize and generate an electrospray. A method and system are disclosed to generate multiple electrospray plumes from a single fluid stream that provides an ion intensity as measured by a mass spectrometer that is approximately proportional to the number of electrospray plumes formed for analytes contained within the fluid. A plurality of electrospray nozzle devices can be used in the form of an array of miniaturized nozzles for the purpose of generating multiple electrospray plumes from multiple nozzles for the same fluid stream. This invention dramatically increases the sensitivity of microchip electrospray devices compared to prior disclosed systems and methods.

[0001] This application claims the benefit of U.S. Provisional PatentApplication Serial No. 60/173,674, filed Dec. 30, 1999, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to an integratedminiaturized fluidic system fabricated using Micro-ElectroMechanicalSystem (MEMS) technology, particularly to an integrated monolithicmicrofabricated device capable of generating multiple sprays from asingle fluid stream.

BACKGROUND OF THE INVENTION

[0003] New trends in drug discovery and development are creating newdemands on analytical techniques. For example, combinatorial chemistryis often employed to discover new lead compounds, or to createvariations of a lead compound. Combinatorial chemistry techniques cangenerate thousands of compounds (combinatorial libraries) in arelatively short time (on the order of days to weeks). Testing such alarge number of compounds for biological activity in a timely andefficient manner requires high-throughput screening methods which allowrapid evaluation of the characteristics of each candidate compound.

[0004] The quality of the combinatorial library and the compoundscontained therein is used to assess the validity of the biologicalscreening data. Confirmation that the correct molecular weight isidentified for each compound or a statistically relevant number ofcompounds along with a measure of compound purity are two importantmeasures of the quality of a combinatorial library. Compounds can beanalytically characterized by removing a portion of solution from eachwell and injecting the contents into a separation device such as liquidchromatography or capillary electrophoresis instrument coupled to a massspectrometer.

[0005] Development of viable screening methods for these new targetswill often depend on the availability of rapid separation and analysistechniques for analyzing the results of assays. For example, an assayfor potential toxic metabolites of a candidate drug would need toidentify both the candidate drug and the metabolites of that candidate.An understanding of how a new compound is absorbed in the body and howit is metabolized can enable prediction of the likelihood for anincreased therapeutic effect or lack thereof.

[0006] Given the enormous number of new compounds that are beinggenerated daily, an improved system for identifying molecules ofpotential therapeutic value for drug discovery is also criticallyneeded. Accordingly, there is a critical need for high-throughputscreening and identification of compound-target reactions in order toidentify potential drug candidates.

[0007] Liquid chromatography (LC) is a well-established analyticalmethod for separating components of a fluid for subsequent analysisand/or identification. Traditionally, liquid chromatography utilizes aseparation column, such as a cylindrical tube with dimensions 4.6 mminner diameter by 25 cm length, filled with tightly packed particles of5 μm diameter. More recently, particles of 3 μm diameter are being usedin shorter length columns. The small particle size provides a largesurface area that can be modified with various chemistries creating astationary phase. A liquid eluent is pumped through the LC column at anoptimized flow rate based on the column dimensions and particle size.This liquid eluent is referred to as the mobile phase. A volume ofsample is injected into the mobile phase prior to the LC column. Theanalytes in the sample interact with the stationary phase based on thepartition coefficients for each of the analytes. The partitioncoefficient is defined as the ratio of the time an analyte spendsinteracting with the stationary phase to the time spent interacting withthe mobile phase. The longer an analyte interacts with the stationaryphase, the higher the partition coefficient and the longer the analyteis retained on the LC column. The diffusion rate for an analyte througha mobile phase (mobile-phase mass transfer) also affects the partitioncoefficient. The mobile-phase mass transfer can be rate limiting in theperformance of the separation column when it is greater than 2 μm (Knox,J. H. J. J. Chromatogr. Sci. 18:453-461 (1980)). Increases inchromatographic separation are achieved when using a smaller particlesize as the stationary phase support.

[0008] The purpose of the LC column is to separate analytes such that aunique response for each analyte from a chosen detector can be acquiredfor a quantitative or qualitative measurement. The ability of a LCcolumn to generate a separation is determined by the dimensions of thecolumn and the particle size supporting the stationary phase. A measureof the ability of LC columns to separate a given analyte is referred toas the theoretical plate number N. The retention time of an analyte canbe adjusted by varying the mobile phase composition and the partitioncoefficient for an analyte. Experimentation and a fundamentalunderstanding of the partition coefficient for a given analyte determinewhich stationary phase is chosen.

[0009] To increase the throughput of LC analyses requires a reduction inthe dimensions of the LC column and the stationary phase particledimensions. Reducing the length of the LC column from 25 cm to 5 cm willresult in a factor of 5 decrease in the retention time for an analyte.At the same time, the theoretical plates are reduced 5-fold. To maintainthe theoretical plates of a 25 cm length column packed with 5 μmparticles, a 5 cm column would need to be packed with 1 μm particles.However, the use of such small particles results in many technicalchallenges.

[0010] One of these technical challenges is the backpressure resultingfrom pushing the mobile phase through each of these columns. Thebackpressure is a measure of the pressure generated in a separationcolumn due to pumping a mobile phase at a given flow rate through the LCcolumn. For example, the typical backpressure of a 4.6 mm inner diameterby 25 cm length column packed with 5 μm particles generates abackpressure of 100 bar at a flow rate of 1.0 mL/min. A 5 cm columnpacked with 1 μm particles generates a back pressure 5 times greaterthan a 25 cm column packed with 5 μm particles. Most commerciallyavailable LC pumps are limited to operating pressures less than 400 barand thus using an LC column with these small particles is not feasible.

[0011] Detection of analytes separated on an LC column has traditionallybeen accomplished by use of spectroscopic detectors. Spectroscopicdetectors rely on a change in refractive index, ultraviolet and/orvisible light absorption, or fluorescence after excitation with asuitable wavelength to detect the separated components. Additionally,the effluent from an LC column may be nebulized to generate an aerosolwhich is sprayed into a chamber to measure the light scatteringproperties of the analytes eluting from the column. Alternatively, theseparated components may be passed from the liquid chromatography columninto other types of analytical instruments for analysis. The volume fromthe LC column to the detector is minimized in order to maintain theseparation efficiency and analysis sensitivity. All system volume notdirectly resulting from the separation column is referred to as the deadvolume or extra-column volume.

[0012] The miniaturization of liquid separation techniques to thenano-scale involves small column internal diameters (<100 μm i.d.) andlow mobile phase flow rates (<300 nL/min). Currently, techniques such ascapillary zone electrophoresis (CZE), nano-LC, open tubular liquidchromatography (OTLC), and capillary electrochromatography (CEC) offernumerous advantages over conventional scale high performance liquidchromatography (HPLC). These advantages include higher separationefficiencies, high-speed separations, analysis of low volume samples,and the coupling of 2-dimensional techniques. One challenge to usingminiaturized separation techniques is detection of the small peakvolumes and a limited number of detectors that can accommodate thesesmall volumes. However, coupling of low flow rate liquid separationtechniques to electrospray mass spectrometry results in a combination oftechniques that are well suited as demonstrated in J. N. Alexander IV,et al., Rapid Commun. Mass Spectrom. 12:1187-91 (1998). The process ofelectrospray at flow rates on the order of nanoliters (“nL”) per minutehas been referred to as “nanoelectrospray”.

[0013] Capillary electrophoresis is a technique that utilizes theelectrophoretic nature of molecules and/or the electroosmotic flow offluids in small capillary tubes to separate components of a fluid.Typically, a fused silica capillary of 100 μm inner diameter or less isfilled with a buffer solution containing an electrolyte. Each end of thecapillary is placed in a separate fluidic reservoir containing a bufferelectrolyte. A potential voltage is placed in one of the bufferreservoirs and a second potential voltage is placed in the other bufferreservoir. Positively and negatively charged species will migrate inopposite directions through the capillary under the influence of theelectric field established by the two potential voltages applied to thebuffer reservoirs. Electroosmotic flow is defined as the fluid flowalong the walls of a capillary due to the migration of charged speciesfrom the buffer solution under the influence of the applied electricfield. Some molecules exist as charged species when in solution and willmigrate through the capillary based on the charge-to-mass ratio of themolecular species. This migration is defined as electrophoreticmobility. The electroosmotic flow and the electrophoretic mobility ofeach component of a fluid determine the overall migration for eachfluidic component. The fluid flow profile resulting from electroosmoticflow is flat due to the reduction in frictional drag along the walls ofthe separation channel. This results in improved separation efficiencycompared to liquid chromatography where the flow profile is parabolicresulting from pressure driven flow.

[0014] Capillary electrochromatography is a hybrid technique thatutilizes the electrically driven flow characteristics of electrophoreticseparation methods within capillary columns packed with a solidstationary phase typical of liquid chromatography. It couples theseparation power of reversed-phase liquid chromatography with the highefficiencies of capillary electrophoresis. Higher efficiencies areobtainable for capillary electrochromatography separations over liquidchromatography, because the flow profile resulting from electroosmoticflow is flat due to the reduction in frictional drag along the walls ofthe separation channel when compared to the parabolic flow profileresulting from pressure driven flows. Furthermore, smaller particlesizes can be used in capillary electrochromatography than in liquidchromatography, because no backpressure is generated by electroosmoticflow. In contrast to electrophoresis, capillary electrochromatography iscapable of separating neutral molecules due to analyte partitioningbetween the stationary and mobile phases of the column particles using aliquid chromatography separation mechanism.

[0015] Microchip-based separation devices have been developed for rapidanalysis of large numbers of samples. Compared to other conventionalseparation devices, these microchip-based separation devices have highersample throughput, reduced sample and reagent consumption, and reducedchemical waste. The liquid flow rates for microchip-based separationdevices range from approximately 1-300 nanoliters per minute for mostapplications. Examples of microchip-based separation devices includethose for capillary electrophoresis (“CE”), capillaryelectrochromatography (“CEC”) and high-performance liquid chromatography(“HPLC”) include Harrison et al., Science 261:859-97 (1993); Jacobson etal., Anal. Chem. 66:1114-18 (1994), Jacobson et al., Anal. Chem.66:2369-73 (1994), Kutter et al., Anal. Chem. 69:5165-71 (1997) and Heet al., Anal. Chem. 70:3790-97 (1998). Such separation devices arecapable of fast analyses and provide improved precision and reliabilitycompared to other conventional analytical instruments.

[0016] The work of He et al., Anal. Chem. 70:3790-97 (1998) demonstratessome of the types of structures that can be fabricated in a glasssubstrate. This work shows that co-located monolithic support structures(or posts) can be etched reproducibly in a glass substrate usingreactive ion etching (RIE) techniques. Currently, anisotropic RIEtechniques for glass substrates are limited to etching features that are20 μm or less in depth. This work shows rectangular 5 μm by 5 μm widthby 10 μm in depth posts and stated that deeper structures were difficultto achieve. The posts are also separated by 1.5 μm. The posts supportsthe stationary phase just as with the particles in LC and CEC columns.An advantage to the posts over conventional LC and CEC is that thestationary phase support structures are monolithic with the substrateand therefore, immobile.

[0017] He et. al., also describes the importance of maintaining aconstant cross-sectional area across the entire length of the separationchannel. Large variations in the cross-sectional area can createpressure drops in pressure driven flow systems. In electrokineticallydriven flow systems, large variations in the cross-sectional area alongthe length of a separation channel can create flow restrictions thatresult in bubble formation in the separation channel. Since the fluidflowing through the separation channel functions as the source andcarrier of the mobile solvated ions, formation of a bubble in aseparation channel will result in the disruption of the electroosmoticflow.

[0018] Electrospray ionization provides for the atmospheric pressureionization of a liquid sample. The electrospray process createshighly-charged droplets that, under evaporation, create ionsrepresentative of the species contained in the solution. An ion-samplingorifice of a mass spectrometer may be used to sample these gas phaseions for mass analysis. When a positive voltage is applied to the tip ofthe capillary relative to an extracting electrode, such as one providedat the ion-sampling orifice of a mass spectrometer, the electric fieldcauses positively-charged ions in the fluid to migrate to the surface ofthe fluid at the tip of the capillary. When a negative voltage isapplied to the tip of the capillary relative to an extracting electrode,such as one provided at the ion-sampling orifice to the massspectrometer, the electric field causes negatively-charged ions in thefluid to migrate to the surface of the fluid at the tip of thecapillary.

[0019] When the repulsion force of the solvated ions exceeds the surfacetension of the fluid being electrosprayed, a volume of the fluid ispulled into the shape of a cone, known as a Taylor cone, which extendsfrom the tip of the capillary. A liquid jet extends from the tip of theTaylor cone and becomes unstable and generates charged-droplets. Thesesmall charged droplets are drawn toward the extracting electrode. Thesmall droplets are highly-charged and solvent evaporation from thedroplets results in the excess charge in the droplet residing on theanalyte molecules in the electrosprayed fluid. The charged molecules orions are drawn through the ion-sampling orifice of the mass spectrometerfor mass analysis. This phenomenon has been described, for example, byDole et al., Chem. Phys. 49:2240 (1968) and Yamashita et al., J. Phys.Chem. 88:4451 (1984). The potential voltage (“V”) required to initiatean electrospray is dependent on the surface tension of the solution asdescribed by, for example, Smith, IEEE Trans. Ind. Appl. 1986,IA-22:527-35 (1986). Typically, the electric field is on the order ofapproximately 10⁶ V/m. The physical size of the capillary and the fluidsurface tension determines the density of electric field lines necessaryto initiate electrospray.

[0020] When the repulsion force of the solvated ions is not sufficientto overcome the surface tension of the fluid exiting the tip of thecapillary, large poorly charged droplets are formed. Fluid droplets areproduced when the electrical potential difference applied between aconductive or partly conductive fluid exiting a capillary and anelectrode is not sufficient to overcome the fluid surface tension toform a Taylor cone.

[0021]Electrospray Ionization Mass Spectrometry: Fundamentals,Instrumentation, and Applications, edited by R. B. Cole, ISBN0-471-14564-5, John Wiley & Sons, Inc., New York summarizes much of thefundamental studies of electrospray. Several mathematical models havebeen generated to explain the principals governing electrospray.Equation 1 defines the electric field E_(c) at the tip of a capillary ofradius r_(c) with an applied voltage V_(c) at a distance d from acounter electrode held at ground potential: $\begin{matrix}{E_{c} = \frac{2V_{c}}{r_{c}{\ln \left( {4{d/r_{c}}} \right)}}} & (1)\end{matrix}$

[0022] The electric field E_(on) required for the formation of a Taylorcone and liquid jet of a fluid flowing to the tip of this capillary isapproximated as: $\begin{matrix}{E_{on} \approx \left( \frac{2{\gamma cos\theta}}{ɛ_{o}r_{c}} \right)^{1/2}} & (2)\end{matrix}$

[0023] where γ is the surface tension of the fluid, θ is the half-angleof the Taylor cone and ε₀ is the permittivity of vacuum. Equation 3 isderived by combining equations 1 and 2 and approximates the onsetvoltage V_(on) required to initiate an electrospray of a fluid from acapillary: $\begin{matrix}{V_{on} \approx {\left( \frac{r_{c}{\gamma cos}\quad \theta}{2ɛ_{0}} \right)^{1/2}{\ln \left( {4{d/r_{c}}} \right)}}} & (3)\end{matrix}$

[0024] As can be seen by examination of equation 3, the required onsetvoltage is more dependent on the capillary radius than the distance fromthe counter-electrode.

[0025] It would be desirable to define an electrospray device that couldform a stable electrospray of all fluids commonly used in CE, CEC, andLC. The surface tension of solvents commonly used as the mobile phasefor these separations range from 100% aqueous (γ=0.073 N/m) to 100%methanol (γ=0.0226 N/m). As the surface tension of the electrosprayfluid increases, a higher onset voltage is required to initiate anelectrospray for a fixed capillary diameter. As an example, a capillarywith a tip diameter of 14 μm is required to electrospray 100% aqueoussolutions with an onset voltage of 1000 V. The work of M. S. Wilm etal., Int. J. Mass Spectrom. Ion Processes 136:167-80 (1994), firstdemonstrates nanoelectrospray from a fused-silica capillary pulled to anouter diameter of 5 μm at a flow rate of 25 nL/min. Specifically, ananoelectrospray at 25 nL/min was achieved from a 2 μm inner diameterand 5 μm outer diameter pulled fused-silica capillary with 600-700 V ata distance of 1-2 mm from the ion-sampling orifice of an electrosprayequipped mass spectrometer.

[0026] Electrospray in front of an ion-sampling orifice of an API massspectrometer produces a quantitative response from the mass spectrometerdetector due to the analyte molecules present in the liquid flowing fromthe capillary. One advantage of electrospray is that the response for ananalyte measured by the mass spectrometer detector is dependent on theconcentration of the analyte in the fluid and independent of the fluidflow rate. The response of an analyte in solution at a givenconcentration would be comparable using electrospray combined with massspectrometry at a flow rate of 100 μL/min compared to a flow rate of 100nL/min. D. C. Gale et al., Rapid Commun. Mass Spectrom. 7:1017 (1993)demonstrate that higher electrospray sensitivity is achieved at lowerflow rates due to increased analyte ionization efficiency. Thus byperforming electrospray on a fluid at flow rates in the nanoliter perminute range provides the best sensitivity for an analyte containedwithin the fluid when combined with mass spectrometry.

[0027] Thus, it is desirable to provide an electrospray device forintegration of microchip-based separation devices with API-MSinstruments. This integration places a restriction on the capillary tipdefining a nozzle on a microchip. This nozzle will, in all embodiments,exist in a planar or near planar geometry with respect to the substratedefining the separation device and/or the electrospray device. When thisco-planar or near planar geometry exists, the electric field linesemanating from the tip of the nozzle will not be enhanced if theelectric field around the nozzle is not defined and controlled and,therefore, an electrospray is only achievable with the application ofrelatively high voltages applied to the fluid.

[0028] Attempts have been made to manufacture an electrospray device formicrochip-based separations. Ramsey et al., Anal. Chem. 69:1174-78(1997) describes a microchip-based separations device coupled with anelectrospray mass spectrometer. Previous work from this research groupincluding Jacobson et al., Anal. Chem. 66:1114-18 (1994) and Jacobson etal., Anal. Chem. 66:2369-73 (1994) demonstrate impressive separationsusing on-chip fluorescence detection. This more recent work demonstratesnanoelectrospray at 90 nL/min from the edge of a planar glass microchip.The microchip-based separation channel has dimensions of 10 μm deep, 60μm wide, and 33 mm in length. Electroosmotic flow is used to generatefluid flow at 90 nL/min. Application of 4,800 V to the fluid exiting theseparation channel on the edge of the microchip at a distance of 3-5 mmfrom the ion-sampling orifice of an API mass spectrometer generates anelectrospray. Approximately 12 nL of the sample fluid collects at theedge of the microchip before the formation of a Taylor cone and stablenanoelectrospray from the edge of the microchip. The volume of thismicrochip-based separation channel is 19.8 nL. Nanoelectrospray from theedge of this microchip device after capillary electrophoresis orcapillary electrochromatography separation is rendered impractical sincethis system has a dead-volume approaching 60% of the column (channel)volume. Furthermore, because this device provides a flat surface, and,thus, a relatively small amount of physical asperity for the formationof the electrospray, the device requires an impractically high voltageto overcome the fluid surface tension to initiate an electrospray.

[0029] Xue, Q. et al., Anal. Chem. 69:426-30 (1997) also describes astable nanoelectrospray from the edge of a planar glass microchip with aclosed channel 25 μm deep, 60 μm wide, and 35-50 mm in length. Anelectrospray is formed by applying 4,200 V to the fluid exiting theseparation channel on the edge of the microchip at a distance of 3-8 mmfrom the ion-sampling orifice of an API mass spectrometer. A syringepump is utilized to deliver the sample fluid to the glass microchip at aflow rate of 100 to 200 nL/min. The edge of the glass microchip istreated with a hydrophobic coating to alleviate some of the difficultiesassociated with nanoelectrospray from a flat surface that slightlyimproves the stability of the nanoelectrospray. Nevertheless, the volumeof the Taylor cone on the edge of the microchip is too large relative tothe volume of the separation channel, making this method of electrospraydirectly from the edge of a microchip impracticable when combined with achromatographic separation device.

[0030] T. D. Lee et. al., 1997 International Conference on Solid-StateSensors and Actuators Chicago, pp. 927-30 (Jun. 16-19, 1997) describes amulti-step process to generate a nozzle on the edge of a siliconmicrochip 1-3 μm in diameter or width and 40 μm in length and applying4,000 V to the entire microchip at a distance of 0.25-0.4 mm from theion-sampling orifice of an API mass spectrometer. Because a relativelyhigh voltage is required to form an electrospray with the nozzlepositioned in very close proximity to the mass spectrometer ion-samplingorifice, this device produces an inefficient electrospray that does notallow for sufficient droplet evaporation before the ions enter theorifice. The extension of the nozzle from the edge of the microchip alsoexposes the nozzle to accidental breakage. More recently, T. D. Leeet.al., in 1999 Twelfth IEEE International Micro Electro MechanicalSystems Conference (Jan. 17-21, 1999), presented this same concept wherethe electrospray component was fabricated to extend 2.5 mm beyond theedge of the microchip to overcome this phenomenon of poor electric fieldcontrol within the proximity of a surface.

[0031] Thus, it is also desirable to provide an electrospray device withcontrollable spraying and a method for producing such a device that iseasily reproducible and manufacturable in high volumes.

[0032] U.S. Pat. No. 5,501,893 to Laermer et. al., reports a method ofanisotropic plasma etching of silicon (Bosch process) that provides amethod of producing deep vertical structures that is easily reproducibleand controllable. This method of anisotropic plasma etching of siliconincorporates a two step process. Step one is an anisotropic etch stepusing a reactive ion etching (RIE) gas plasma of sulfur hexafluoride(SF₆). Step two is a passivation step that deposits a polymer on thevertical surfaces of the silicon substrate. This polymerizing stepprovides an etch stop on the vertical surface that was exposed in stepone. This two step cycle of etch and passivation is repeated until thedepth of the desired structure is achieved. This method of anisotropicplasma etching provides etch rates over 3 μm/min of silicon depending onthe size of the feature being etched. The process also providesselectivity to etching silicon versus silicon dioxide or resist ofgreater than 100:1 which is important when deep silicon structures aredesired. Laermer et. al., in 1999 Twelfth IEEE International MicroElectro Mechanical Systems Conference (Jan. 17-21, 1999), reportedimprovements to the Bosch process. These improvements include siliconetch rates approaching 10 μm/min, selectivity exceeding 300:1 to silicondioxide masks, and more uniform etch rates for features that vary insize.

[0033] The present invention is directed toward a novel utilization ofthese features to improve the sensitivity of prior disclosedmicrochip-based electrospray systems.

SUMMARY OF THE INVENTION

[0034] The present invention relates to an electrospray device forspraying a fluid which includes an insulating substrate having aninjection surface and an ejection surface opposing the injectionsurface. The substrate is an integral monolith having either a singlespray unit or a plurality of spray units for generating multiple spraysfrom a single fluid stream. Each spray unit includes an entrance orificeon the injection surface; an exit orifice on the ejection surface; achannel extending between the entrance orifice and the exit orifice; anda recess surrounding the exit orifice and positioned between theinjection surface and the ejection surface. The entrance orifices foreach of the plurality of spray units are in fluid communication with oneanother and each spray unit generates an electrospray plume of thefluid. The electrospray device also includes an electric fieldgenerating source positioned to define an electric field surrounding theexit orifice. In one embodiment, the electric field generating sourceincludes a first electrode attached to the substrate to impart a firstpotential to the substrate and a second electrode to impart a secondpotential. The first and the second electrodes are positioned to definean electric field surrounding the exit orifice. This device can beoperated to generate multiple electrospray plumes of fluid from eachspray unit, to generate a single combined electrospray plume of fluidfrom a plurality of spray units, and to generate multiple electrosprayplumes of fluid from a plurality of spray units. The device can also beused in conjunction with a system for processing an electrospray offluid, a method of generating an electrospray of fluid, a method of massspectrometric analysis, and a method of liquid chromatographic analysis.

[0035] Another aspect of the present invention is directed to anelectrospray system for generating multiple sprays from a single fluidstream. The system includes an array of a plurality of the aboveelectrospray devices. The electrospray devices can be provided in thearray at a device density exceeding about 5 devices/cm², about 16devices/cm², about 30 devices/cm², or about 81 devices/cm². Theelectrospray devices can also be provided in the array at a devicedensity of from about 30 devices/cm² to about 100 devices/cm².

[0036] Another aspect of the present invention is directed to an arrayof a plurality of the above electrospray devices for generating multiplesprays from a single fluid stream. The electrospray devices can beprovided in an array wherein the spacing on the ejection surface betweenadjacent devices is about 9 mm or less, about 4.5 mm or less, about 2.2mm or less, about 1.1 mm or less, about 0.56 mm or less, or about 0.28mm or less, respectively.

[0037] Another aspect of the present invention is directed to a methodof generating an electrospray wherein an electrospray device is providedfor spraying a fluid. The electospray device includes a substrate havingan injection surface and an ejection surface opposing the injectionsurface. The substrate is an integral monolith which includes anentrance orifice on the injection surface; an exit orifice on theejection surface; a channel extending between the entrance orifice andthe exit orifice; and a recess surrounding the exit orifice andpositioned between the injection surface and the ejection surface. Themethod can be performed to generate multiple electrospray plumes offluid from each spray unit, to generate a single combined electrosprayplume of fluid from a plurality of spray units, and to generate multipleelectrospray plumes of fluid from a plurality of spray units. Theelectrospray device also includes an electric field generating sourcepositioned to define an electric field surrounding the exit orifice. Inone embodiment, the electric field generating source includes a firstelectrode attached to the substrate to impart a first potential to thesubstrate and a second electrode to impart a second potential. The firstand the second electrodes are positioned to define an electric fieldsurrounding the exit orifice. Analyte from a fluid sample is depositedon the injection surface and then eluted with an eluting fluid. Theeluting fluid containing analyte is passed into the entrance orificethrough the channel and through the exit orifice. A first potential isapplied to the first electrode and a second potential is applied to thefluid through the second electrode. The first and second potentials areselected such that fluid discharged from the exit orifice of each of thespray units forms an electrospray.

[0038] Another aspect of the present invention is directed to a methodof producing an electrospray device which includes providing a substratehaving opposed first and second surfaces, each coated with a photoresistover an etch-resistant material. The photoresist on the first surface isexposed to an image to form a pattern in the form of at least one ringon the first surface. The photoresist on the first surface which isoutside and inside the at least one ring is then removed to form anannular portion. The etch-resistant material is removed from the firstsurface of the substrate where the photoresist is removed to form holesin the etch-resistant material. Photoresist remaining on the firstsurface is then optionally removed. The first surface is then coatedwith a second coating of photoresist. The second coating of photoresistwithin the at least one ring is exposed to an image and removed to format least one hole. The material from the substrate coincident with theat least one hole in the second layer of photoresist on the firstsurface is removed to form at least one passage extending through thesecond layer of photoresist on the first surface and into the substrate.Photoresist from the first surface is then removed. An etch-resistantlayer is applied to all exposed surfaces on the first surface side ofthe substrate. The etch-resistant layer from the first surface that isaround the at least one ring and the material from the substrate aroundthe at least one ring are removed to define at least one nozzle on thefirst surface. The photoresist on the second surface is then exposed toan image to form a pattern circumscribing extensions of the at least onehole formed in the etch-resistant material of the first surface. Theetch-resistant material on the second surface is then removed where thepattern is. Material is removed from the substrate coincident with wherethe pattern in the photoresist on the second surface has been removed toform a reservoir extending into the substrate to the extent needed tojoin the reservoir and the at least one passage. An etch-resistantmaterial is then applied to all exposed surfaces of the substrate toform the electrospray device. The method further includes the step ofapplying a silicon nitride layer over all surfaces after theetch-resistant material is applied to all exposed surfaces of thesubstrate.

[0039] Another aspect of the present invention is directed anothermethod of producing an electrospray device including providing asubstrate having opposed first and second surfaces, the first sidecoated with a photoresist over an etch-resistant material. Thephotoresist on the first surface is exposed to an image to form apattern in the form of at least one ring on the first surface. Theexposed photoresist is removed on the first surface which is outside andinside the at least one ring leaving the unexposed photoresist. Theetch-resistant material is removed from the first surface of thesubstrate where the exposed photoresist was removed to form holes in theetch-resistant material. Photoresist is removed from the first surface.Photoresist is provided over an etch-resistant material on the secondsurface and exposed to an image to form a pattern circumscribingextensions of the at least one ring formed in the etch-resistantmaterial of the first surface. The exposed photoresist on the secondsurface is removed. The etch-resistant material on the second surface isremoved coincident with where the photoresist was removed. Material isremoved from the substrate coincident with where the etch-resistantmaterial on the second surface was removed to form a reservoir extendinginto the substrate. The remaining photoresist on the second surface isremoved. The second surface is coated with an etch-resistant material.The first surface is coated with a second coating of photoresist. Thesecond coating of photoresist within the at least one ring is exposed toan image. The exposed second coating of photoresist is removed fromwithin the at least one ring to form at least one hole. Material isremoved from the substrate coincident with the at least one hole in thesecond layer of photoresist on the first surface to form at least onepassage extending through the second layer of photoresist on the firstsurface and into substrate to the extent needed to reach theetch-resistant material coating the reservoir. Photoresist from thefirst surface is removed. Material is removed from the substrate exposedby the removed etch-resistant layer around the at least one ring todefine at least one nozzle on the first surface. The etch-resistantmaterial coating the reservoir is removed from the substrate. An etchresistant material is applied to coat all exposed surfaces of thesubstrate to form the electrospray device.

[0040] The electrospray device of the present invention can generatemultiple electrospray plumes from a single fluid stream and besimultaneously combined with mass spectrometry. Each electrospray plumegenerates a signal for an analyte contained within a fluid that isproportional to that analytes concentration. When multiple electrosprayplumes are generated from one nozzle, the ion intensity for a givenanalyte will increase with the number of electrospray plumes emanatingfrom that nozzle as measured by the mass spectrometer. When multiplenozzle arrays generate one or more electrospray plumes, the ionintensity will increase with the number of nozzles times the number ofelectrospray plumes emanating from the nozzle arrays.

[0041] The present invention achieves a significant advantage in termsof high-sensitivity analysis of analytes by electrospray massspectrometry. A method of control of the electric field around closelypositioned electrospray nozzles provides a method of generating multipleelectrospray plumes from closely positioned nozzles in a well-controlledprocess. An array of electrospray nozzles is disclosed for generation ofmultiple electrospray plumes of a solution for purpose of generating anion response as measured by a mass spectrometer that increases with thetotal number of generated electrospray plumes. The present inventionachieves a significant advantage in comparison to prior disclosedelectrospray systems and methods for combination with microfluidicchip-based devices incorporating a single nozzle forming a singleelectrospray.

[0042] The electrospray device of the present invention generallyincludes a silicon substrate material defining a channel between anentrance orifice on an injection surface and a nozzle on an ejectionsurface (the major surface) such that the electrospray generated by thedevice is generally perpendicular to the ejection surface. The nozzlehas an inner and an outer diameter and is defined by an annular portionrecessed from the ejection surface. The recessed annular region extendsradially from the outer diameter. The tip of the nozzle is co-planar orlevel with and does not extend beyond the ejection surface. Thus, thenozzle is protected against accidental breakage. The nozzle, thechannel, and the recessed annular region are etched from the siliconsubstrate by deep reactive-ion etching and other standard semiconductorprocessing techniques.

[0043] All surfaces of the silicon substrate preferably have insulatinglayers thereon to electrically isolate the liquid sample from thesubstrate and the ejection and injection surfaces from each other suchthat different potential voltages may be individually applied to eachsurface, the silicon substrate and the liquid sample. The insulatinglayer generally constitutes a silicon dioxide layer combined with asilicon nitride layer. The silicon nitride layer provides a moisturebarrier against water and ions from penetrating through to the substratethus preventing electrical breakdown between a fluid moving in thechannel and the substrate. The electrospray apparatus preferablyincludes at least one controlling electrode electrically contacting thesubstrate for the application of an electric potential to the substrate.

[0044] Preferably, the nozzle, channel and recess are etched from thesilicon substrate by reactive-ion etching and other standardsemiconductor processing techniques. The injection-side features,through-substrate fluid channel, ejection-side features, and controllingelectrodes are formed monolithically from a monocrystalline siliconsubstrate—i.e., they are formed during the course of and as a result ofa fabrication sequence that requires no manipulation or assembly ofseparate components.

[0045] Because the electrospray device is manufactured usingreactive-ion etching and other standard semiconductor processingtechniques, the dimensions of such a device nozzle can be very small,for example, as small as 2 μm inner diameter and 5 μm outer diameter.Thus, a through-substrate fluid channel having, for example, 5 μm innerdiameter and a substrate thickness of 250 μm only has a volume of 4.9 pL(“picoliters”). The micrometer-scale dimensions of the electrospraydevice minimize the dead volume and thereby increase efficiency andanalysis sensitivity when combined with a separation device.

[0046] The electrospray device of the present invention provides for theefficient and effective formation of an electrospray. By providing anelectrospray surface (i.e., the tip of the nozzle) from which the fluidis ejected with dimensions on the order of micrometers, the devicelimits the voltage required to generate a Taylor cone and subsequentelectrospray. The nozzle of the electrospray device provides thephysical asperity on the order of micrometers on which a large electricfield is concentrated. Further, the nozzle of the electrospray devicecontains a thin region of conductive silicon insulated from a fluidmoving through the nozzle by the insulating silicon dioxide and siliconnitride layers. The fluid and substrate voltages and the thickness ofthe insulating layers separating the silicon substrate from the fluiddetermine the electric field at the tip of the nozzle. Additionalelectrode(s) on the ejection surface to which electric potential(s) maybe applied and controlled independent of the electric potentials of thefluid and the substrate may be incorporated in order to advantageouslymodify and optimize the electric field in order to focus the gas phaseions produced by the electrospray.

[0047] The microchip-based electrospray device of the present inventionprovides minimal extra-column dispersion as a result of a reduction inthe extra-column volume and provides efficient, reproducible, reliableand rugged formation of an electrospray. This electrospray device isperfectly suited as a means of electrospray of fluids frommicrochip-based separation devices. The design of this electrospraydevice is also robust such that the device can be readily mass-producedin a cost-effective, high-yielding process.

[0048] The electrospray device may be interfaced to or integrateddownstream from a sampling device, depending on the particularapplication. For example, the analyte may be electrosprayed onto asurface to coat that surface or into another device for purposes ofconveyance, analysis, and/or synthesis. As described previously, highlycharged droplets are formed at atmospheric pressure by the electrospraydevice from nanoliter-scale volumes of an analyte. The highly chargeddroplets produce gas-phase ions upon sufficient evaporation of solventmolecules which may be sampled, for example, through an ion-samplingorifice of an atmospheric pressure ionization mass spectrometer(“API-MS”) for analysis of the electrosprayed fluid.

[0049] A multi-system chip thus provides a rapid sequential chemicalanalysis system fabricated using Micro-ElectroMechanical System (“MEMS”)technology. The multi-system chip enables automated, sequentialseparation and injection of a multiplicity of samples, resulting insignificantly greater analysis throughput and utilization of the massspectrometer instrument for high-throughput detection of compounds fordrug discovery.

[0050] Another aspect of the present invention provides a siliconmicrochip-based electrospray device for producing electrospray of aliquid sample. The electrospray device may be interfaced downstream toan atmospheric pressure ionization mass spectrometer (“API-MS”) foranalysis of the electrosprayed fluid.

[0051] The use of multiple nozzles for electrospray of fluid from thesame fluid stream extends the useful flow rate range of microchip-basedelectrospray devices. Thus, fluids may be introduced to the multipleelectrospray device at higher flow rates as the total fluid flow issplit between all of the nozzles. For example, by using 10 nozzles perfluid channel, the total flow can be 10 times higher than when usingonly one nozzle per fluid channel. Likewise, by using 100 nozzles perfluid channel, the total flow can be 100 times higher than when usingonly one nozzle per fluid channel. The fabrication methods used to formthese electrospray nozzles allow for multiple nozzles to be easilycombined with a single fluid stream channel greatly extending the usefulfluid flow rate range and increasing the mass spectral sensitivity formicrofluidic devices.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1A shows a plan view of a one-nozzle electrospray device ofthe present invention.

[0053]FIG. 1B shows a plan view of a two-nozzle electrospray device ofthe present invention.

[0054]FIG. 1C shows a plan view of a three-nozzle electrospray device ofthe present invention.

[0055]FIG. 1D shows a plan view of a fourteen-nozzle electrospray deviceof the present invention.

[0056]FIG. 2A shows a perspective view of a one-nozzle electrospraydevice of the present invention.

[0057]FIG. 2B shows a perspective view of a two-nozzle electrospraydevice of the present invention.

[0058]FIG. 2C shows a perspective view of a three-nozzle electrospraydevice of the present invention.

[0059]FIG. 2D shows a perspective view of a fourteen-nozzle electrospraydevice of the present invention.

[0060]FIG. 3A shows a cross-sectional view of a one-nozzle electrospraydevice of the present invention.

[0061]FIG. 3B shows a cross-sectional view of a two-nozzle electrospraydevice of the present invention.

[0062]FIG. 3C shows a cross-sectional view of a three-nozzleelectrospray device of the present invention.

[0063]FIG. 3D shows a cross-sectional view of a fourteen-nozzleelectrospray device of the present invention.

[0064]FIG. 4 is a perspective view of the injection or reservoir side ofan electrospray device of the present invention.

[0065]FIG. 5A shows a cross-sectional view of a two-nozzle electrospraydevice of the present invention generating one electrospray plume fromeach nozzle.

[0066]FIG. 5B shows a cross-sectional view of a two-nozzle electrospraydevice of the present invention generating two electrospray plumes fromeach nozzle.

[0067]FIG. 6A shows a perspective view of a one-nozzle electrospraydevice of the present invention generating one electrospray plume fromone nozzle.

[0068]FIG. 6B shows a perspective view of a one-nozzle electrospraydevice of the present invention generating two electrospray plumes fromone nozzle.

[0069]FIG. 6C shows a perspective view of a one-nozzle electrospraydevice of the present invention generating three electrospray plumesfrom one nozzle.

[0070]FIG. 6D shows a perspective view of a one-nozzle electrospraydevice of the present invention generating four electrospray plumes fromone nozzle.

[0071]FIG. 7A shows a video capture picture of a microfabricatedelectrospray nozzle generating one electrospray plume from one nozzle.

[0072]FIG. 7B shows a video capture picture of a microfabricatedelectrospray nozzle generating two electrospray plumes from one nozzle.

[0073]FIG. 8A shows the total ion chromatogram (“TIC”) of a solutionundergoing electrospray.

[0074]FIG. 8B shows the mass chromatogram for the protonated analyte atm/z 315. Region 1 is the resulting ion intensity from one electrosprayplume from one nozzle. Region 2 is from two electrospray plumes from onenozzle. Region 3 is from three electrospray plumes from one nozzle.Region 4 is from four electrospray plumes from one nozzle. Region 5 isfrom two electrospray plumes from one nozzle.

[0075]FIG. 9A shows the mass spectrum from Region 1 of FIG. 8B.

[0076]FIG. 9B shows the mass spectrum from Region 2 of FIG. 8B.

[0077]FIG. 9C shows the mass spectrum from Region 3 of FIG. 8B.

[0078]FIG. 9D shows the mass spectrum from Region 4 of FIG. 8B.

[0079]FIG. 10 is a chart of the ion intensity for m/z 315 versus thenumber of electrospray plumes emanating from one nozzle.

[0080]FIG. 11A is a plan view of a two by two array of groups of fournozzles of an electrospray device.

[0081]FIG. 11B is a perspective view of a two by two array of groups offour nozzles taken through a line through one row of nozzles.

[0082]FIG. 11C is a cross-sectional view of a two by two array of groupsof four nozzles of an electrospray device.

[0083]FIG. 12A is a cross-sectional view of a 20 μm diameter nozzle witha nozzle height of 50 μm. The fluid has a voltage of 1000V, substratehas a voltage of zero V and a third electrode (not shown due to thescale of the figure) is located 5 mm from the substrate and has avoltage of zero V. The equipotential field lines are shown in incrementsof 50 V.

[0084]FIG. 12B is an expanded region around the nozzle shown in FIG.12A.

[0085]FIG. 12C is a cross-sectional view of a 20 μm diameter nozzle witha nozzle height of 50 μm. The fluid has a voltage of 1000V, substratehas a voltage of zero V and a third electrode (not shown due to thescale of the figure) is located 5 mm from the substrate and has avoltage of 800 V. The equipotential field lines are shown in incrementsof 50 V.

[0086]FIG. 12D is a cross-sectional view of a 20 μm diameter nozzle witha nozzle height of 50 μm. The fluid has a voltage of 1000V, substratehas a voltage of 800 V and a third electrode (not shown due to the scaleof the figure) is located 5 mm from the substrate and has a voltage ofzero V. The equipotential field lines are shown in increments of 50 V.

[0087] FIGS. 13A-13C are cross-sectional views of an electrospray deviceof the present invention illustrating the transfer of a discreet samplequantity to a reservoir contained on the substrate surface.

[0088]FIG. 13D is a cross-sectional view of an electrospray device ofthe present invention illustrating the evaporation of the solutionleaving an analyte contained within the fluid on the surface of thereservoir.

[0089]FIG. 13E is a cross-sectional view of an electrospray device ofthe present invention illustrating a fluidic probe sealed against theinjection surface delivering a reconstitution fluid to redissolve theanalyte for electrospray mass spectrometry analysis.

[0090]FIG. 14A is a plan view of mask one of an electrospray device.

[0091]FIG. 14B is a cross-sectional view of a silicon substrate 200showing silicon dioxide layers 210 and 212 and photoresist layer 208.

[0092]FIG. 14C is a cross-sectional view of a silicon substrate 200showing removal of photoresist layer 208 to form a pattern of 204 and206 in the photoresist.

[0093]FIG. 14D is a cross-sectional view of a silicon substrate 200showing removal of silicon dioxide 210 from the regions 212 and 214 toexpose the silicon substrate in these regions to form a pattern of 204and 206 in the silicon dioxide 210.

[0094]FIG. 14E is a cross-sectional view of a silicon substrate 200showing removal of photoresist 208.

[0095]FIG. 15A is a plan view of mask two of an electrospray device.

[0096]FIG. 15B is a cross-sectional view of a silicon substrate 200 ofFIG. 14E with a new layer of photoresist 208′.

[0097]FIG. 15C is a cross-sectional view of a silicon substrate 200showing of removal of photoresist layer 208′ to form a pattern of 204 inthe photoresist and exposing the silicon substrate 218.

[0098]FIG. 15D is a cross-sectional view of a silicon substrate 200showing the removal of silicon substrate material from the region 218 toform a cylinder 224.

[0099]FIG. 15E is a cross-sectional view of a silicon substrate 200showing removal of photoresist 208′.

[0100]FIG. 15F is a cross-sectional view of a silicon substrate 200showing thermal oxidation of the exposed silicon substrate 200 to form alayer of silicon dioxide 226 and 228 on exposed silicon horizontal andvertical surfaces, respectively.

[0101]FIG. 15G is a cross-sectional view of a silicon substrate 200showing selective removal of silicon dioxide 226 from all horizontalsurfaces.

[0102]FIG. 15H is a cross-sectional view of a silicon substrate 200showing removal of silicon substrate 220 to form an annular space 230around the nozzles 232.

[0103]FIG. 16A is a plan view of mask three of an electrospray deviceshowing reservoir 234.

[0104]FIG. 16B is a cross-sectional view of a silicon substrate 200 ofFIG. 15I with a new layer of photoresist 232 on silicon dioxide 212.

[0105]FIG. 16C is a cross-sectional view of a silicon substrate 200showing removal of photoresist layer 232 to form a pattern 234 in thephotoresist exposing silicon dioxide 236.

[0106]FIG. 16D is a cross-sectional view of a silicon substrate 200showing removal of silicon dioxide 236 from region 234 to expose silicon238 in the pattern of 234.

[0107]FIG. 16E is a cross-sectional view of a silicon substrate 200showing removal of silicon 238 from region 234 to form reservoir 240 inthe pattern of 234.

[0108]FIG. 16F is a cross-sectional view of a silicon substrate 200showing removal of photoresist 232.

[0109]FIG. 16G is a cross-sectional view of a silicon substrate 200showing thermal oxidation of the exposed silicon substrate 200 to form alayer of silicon dioxide 242 on all exposed silicon surfaces.

[0110]FIG. 16H is a cross-sectional view of a silicon substrate 200showing low pressure vapor deposition of silicon nitride 244 conformallycoating all surfaces of the electrospray device 300.

[0111]FIG. 16I is a cross-sectional view of a silicon substrate 200showing metal deposition of electrode 246 on silicon substrate 200.

[0112]FIG. 17A is a plan view of mask four of an electrospray device.

[0113]FIG. 17B is a cross-sectional view of a silicon substrate 300showing silicon dioxide layers 310 and 312 and photoresist layer 308.

[0114]FIG. 17C is a cross-sectional view of a silicon substrate 300showing removal of photoresist layer 308 to form a pattern of 304 and306 in the photoresist.

[0115]FIG. 17D is a cross-sectional view of a silicon substrate 300showing removal of silicon dioxide 310 from the regions 318 and 320 toexpose the silicon substrate in these regions to form a pattern of 204and 206 in the silicon dioxide 310.

[0116]FIG. 17E is a cross-sectional view of a silicon substrate 300showing removal of photoresist 308.

[0117]FIG. 18A is a plan view of mask five of an electrospray device.

[0118]FIG. 18B is a cross-sectional view of a silicon substrate 300showing deposition of a film of positive-working photoresist 326 on thesilicon dioxide layer 312.

[0119]FIG. 18C is a cross-sectional view of a silicon substrate 300showing removal of exposed areas 324 of photoresist layer 326.

[0120]FIG. 18D is a cross-sectional view of a silicon substrate 300showing etching of the exposed area 328 of the silicon dioxide layer312.

[0121]FIG. 18E is a cross-sectional view of a silicon substrate 300showing the etching of reservoir 332.

[0122]FIG. 18F is a cross-sectional view of a silicon substrate 300showing removal of the remaining photoresist 326.

[0123]FIG. 18G is a cross-sectional view of a silicon substrate 300showing deposition of the silicon dioxide layer 334.

[0124]FIG. 19A is a plan view of mask six of an electrospray deviceshowing through-wafer channels 304.

[0125]FIG. 19B is a cross-sectional view of a silicon substrate 300showing deposition of a layer of photoresist 308′ on silicon dioxidelayer 310.

[0126]FIG. 19C is a cross-sectional view of a silicon substrate 300showing removal of the exposed area 304 of the photoresist.

[0127]FIG. 19D is a cross-sectional view of a silicon substrate 300showing etching of the through-wafer channels 336.

[0128]FIG. 19E is a cross-sectional view of a silicon substrate 300showing removal of photoresist 308′.

[0129]FIG. 19F is a cross-sectional view of a silicon substrate 300showing removal of silicon substrate 320 to form an annular space 338around the nozzles.

[0130]FIG. 19G is a cross-sectional view of a silicon substrate 300showing removal of silicon dioxide layers 310, 312 and 334.

[0131]FIG. 20A is a cross-sectional view of a silicon substrate 300showing deposition of silicon dioxide layer 342 coating all siliconsurfaces of the electrospray device 300.

[0132]FIG. 20B is a cross-sectional view of a silicon substrate 300showing deposition of silicon nitride layer 344 coating all surfaces ofthe electrospray device 300.

[0133]FIG. 20C is a cross-sectional view of a silicon substrate 300showing metal deposition of electrodes 346 and 348.

[0134]FIGS. 21A and 21B show a perspective view of scanning electronmicrograph images of a multi-nozzle device fabricated in accordance withthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0135] Control of the electric field at the tip of a nozzle is animportant component for successful generation of an electrospray formicrofluidic microchip-based systems. This invention provides sufficientcontrol and definition of the electric field in and around a nozzlemicrofabricated from a monolithic silicon substrate for the formation ofmultiple electrospray plumes from closely positioned nozzles. Thepresent nozzle system is fabricated using Micro-ElectroMechanical System(“MEMS”) fabrication technologies designed to micromachine 3-dimensionalfeatures from a silicon substrate. MEMS technology, in particular, deepreactive ion etching (“DRIE”), enables etching of the small verticalfeatures required for the formation of micrometer dimension surfaces inthe form of a nozzle for successful nanoelectrospray of fluids.Insulating layers of silicon dioxide and silicon nitride are also usedfor independent application of an electric field surrounding the nozzle,preferably by application of a potential voltage to a fluid flowingthrough the silicon device and a potential voltage applied to thesilicon substrate. This independent application of a potential voltageto a fluid exiting the nozzle tip and the silicon substrate creates ahigh electric field, on the order of 10⁸ V/m, at the tip of the nozzle.This high electric field at the nozzle tip causes the formation of aTaylor cone, fluidic jet and highly-charged fluidic dropletscharacteristic of the electrospray of fluids. These two voltages, thefluid voltage and the substrate voltage, control the formation of astable electrospray from this microchip-based electrospray device.

[0136] The electrical properties of silicon and silicon-based materialsare well characterized. The use of silicon dioxide and silicon nitridelayers grown or deposited on the surfaces of a silicon substrate arewell known to provide electrical insulating properties. Incorporatingsilicon dioxide and silicon nitride layers in a monolithic siliconelectrospray device with a defined nozzle provides for the enhancementof an electric field in and around features etched from a monolithicsilicon substrate. This is accomplished by independent application of avoltage to the fluid exiting the nozzle and the region surrounding thenozzle. Silicon dioxide layers may be grown thermally in an oven to adesired thickness. Silicon nitride can be deposited using low pressurechemical vapor deposition (“LPCVD”). Metals may be further vapordeposited on these surfaces to provide for application of a potentialvoltage on the surface of the device. Both silicon dioxide and siliconnitride function as electrical insulators allowing the application of apotential voltage to the substrate that is different than that appliedto the surface of the device. An important feature of a silicon nitridelayer is that it provides a moisture barrier between the siliconsubstrate, silicon dioxide and any fluid sample that comes in contactwith the device. Silicon nitride prevents water and ions from diffusingthrough the silicon dioxide layer to the silicon substrate which maycause an electrical breakdown between the fluid and the siliconsubstrate. Additional layers of silicon dioxide, metals and othermaterials may further be deposited on the silicon nitride layer toprovide chemical functionality to silicon-based devices.

[0137] FIGS. 1A-1D show plan views of 1, 2, 3 and 14 nozzle electrospraydevices, respectively, of the present invention. FIGS. 2A-2D showperspective views of the nozzle side of an electrospray device showing1, 2, 3 and 14 nozzles 232, respectively, etched from the siliconsubstrate 200. FIGS. 3A-3D show cross-sectional views of 1, 2, 3 and 14nozzle electrospray devices, respectively. The nozzle or ejection sideof the device and the reservoir or injection side of the device areconnected by the through-wafer channels 224 thus creating a fluidic paththrough the silicon substrate 200.

[0138] Fluids may be introduced to this microfabricated electrospraydevice by a fluid delivery device such as a probe, conduit, capillary,micropipette, microchip, or the like. The perspective view of FIG. 4shows a probe 252 that moves into contact with the injection orreservoir side of the electrospray device of the present invention. Theprobe can have a disposable tip. This fluid probe has a seal, forexample an o-ring 254, at the tip to form a seal between the probe tipand the injection surface of the substrate 200. FIG. 4 shows an array ofa plurality of electrospray devices fabricated on a monolithicsubstrate. One liquid sample handling device is shown for clarity,however, multiple liquid sampling devices can be utilized to provide oneor more fluid samples to one or more electrospray devices in accordancewith the present invention. The fluid probe and the substrate can bemanipulated in 3-dimensions for staging of, for example, differentdevices in front of a mass spectrometer or other sample detectionapparatus.

[0139] As shown in FIG. 5, to generate an electrospray, fluid may bedelivered to the through-substrate channel 224 of the electrospraydevice 250 by, for example, a capillary 256, micropipette or microchip.The fluid is subjected to a potential voltage, for example, in thecapillary 256 or in the reservoir 242 or via an electrode provided onthe reservoir surface and isolated from the surrounding surface regionand the substrate 200. A potential voltage may also be applied to thesilicon substrate via the electrode 246 on the edge of the siliconsubstrate 200 the magnitude of which is preferably adjustable foroptimization of the electrospray characteristics. The fluid flowsthrough the channel 224 and exits from the nozzle 232 in the form of aTaylor cone 258, liquid jet 260, and very fine, highly charged fluidicdroplets 262. FIG. 5 shows a cross-sectional view of a two-nozzle arrayof the present invention. FIG. 5A shows a cross-sectional view of a 2nozzle electrospray device generating one electrospray plume from eachnozzle for a single fluid stream. FIG. 5B shows a cross-sectional viewof a 2 nozzle electrospray device generating 2 electrospray plumes fromeach nozzle for a single fluid stream.

[0140] The nozzle 232 provides the physical asperity to promote theformation of a Taylor cone 258 and efficient electrospray 262 of a fluid256. The nozzle 232 also forms a continuation of and serves as an exitorifice of the through-wafer channel 224. The recessed annular region230 serves to physically isolate the nozzle 232 from the surface. Thepresent invention allows the optimization of the electric field linesemanating from the fluid 256 exiting the nozzle 232, for example,through independent control of the potential voltage of the fluid 256and the potential voltage of the substrate 200.

[0141] FIGS. 6A-6D illustrate 1, 2, 3 and 4 electrospray plumes,respectively, generated from one nozzle 232. FIGS. 7A-7B show videocapture pictures of a microfabricated electrospray device of the presentinvention generating one electrospray plume from one nozzle and twoelectrospray plumes from one nozzle, respectively. FIG. 8 shows massspectral results acquired from a microfabricated electrospray device ofthe present invention generating from 1 to 4 electrospray plumes from asingle nozzle. The applied fluid potential voltage relative to theapplied substrate potential voltage controls the number of electrosprayplumes generated. FIG. 8A shows the total ion chromatogram (“TIC”) of asolution containing an analyte at a concentration of 5 μM resulting fromelectrospray of the fluid from a microfabricated electrospray device ofthe present invention. The substrate voltage for this example is held atzero V while the fluid voltage is varied to control the number ofelectrospray plumes exiting the nozzle. FIG. 8B shows the selected masschromatogram for the analyte at m/z 315. In this example, Region I hasone electrospray plume exiting the nozzle tip with a fluid voltage of950V. Region II has two electrospray plumes exiting the nozzle tip witha fluid voltage of 1050V. Region III has three electrospray plumesexiting the nozzle tip with a fluid voltage of 1150 V. Region IV hasfour electrospray plumes exiting the nozzle tip with a fluid voltage of1250V. Region V has two electrospray plumes exiting the nozzle tip.

[0142]FIG. 9A shows the mass spectrum resulting from Region I with oneelectrospray plume. FIG. 9B shows the mass spectrum resulting fromRegion II with two electrospray plumes. FIG. 9C shows the mass spectrumresulting from Region III with three electrospray plumes. FIG. 9D showsthe mass spectrum resulting from Region IV with four electrospray plumesexiting the nozzle tip. It is clear from the results that this inventioncan provide an increase in the analyte response measured by a massspectrometer proportional to the number of electrospray plumes exitingthe nozzle tip. FIG. 10 charts the ion intensity for m/z 315 for 1, 2, 3and 4 electrospray plumes exiting the nozzle tip.

[0143] FIGS. 11A-11C illustrate a system having a two by two array ofelectrospray devices. Each device has a group of four electrospraynozzles in fluid communication with one common reservoir containing asingle fluid sample source. Thus, this system can generate multiplesprays for each fluid stream up to four different fluid streams.

[0144] The electric field at the nozzle tip can be simulated usingSIMION™ ion optics software. SIMION™ allows for the simulation ofelectric field lines for a defined array of electrodes. FIG. 12A shows across-sectional view of a 20 μm diameter nozzle 232 with a nozzle heightof 50 μm. A fluid 256 flowing through the nozzle 232 and exiting thenozzle tip in the shape of a hemisphere has a potential voltage of1000V. The substrate 200 has a potential voltage of zero volts. Asimulated third electrode (not shown in the figure due to the scale ofthe drawing) is located 5 mm from the nozzle side of the substrate andhas a potential voltage of zero volts. This third electrode is generallyan ion-sampling orifice of an atmospheric pressure ionization massspectrometer. This simulates the electric field required for theformation of a Taylor cone rather than the electric field required tomaintain an electrospray. FIG. 12A shows the equipotential lines in 50 Vincrements. The closer the equipotential lines are spaced the higher theelectric field. The simulated electric field at the fluid tip with thesedimensions and potential voltages is 8.2×10⁷ V/m. FIG. 12B shows anexpanded region around the nozzle of FIG. 12A to show greater detail ofthe equipotential lines. FIG. 12C shows the equipotential lines aroundthis same nozzle with a fluid potential voltage of 1000V, substratevoltage of zero V and a third electrode voltage of 800 V. The electricfield at the nozzle tip is 8.0×10⁷ V/m indicating that the appliedvoltage of this third electrode has little effect on the electric fieldat the nozzle tip. FIG. 12D shows the electric field lines around thissame nozzle with a fluid potential voltage of 1000V, substrate voltageof 800 V and a third electrode voltage of 0 V. The electric field at thenozzle tip is reduced significantly to a value of 2.2×10⁷ V/m. Thisindicates that very fine control of the electric field at the nozzle tipis achieved with this invention by independent control of the appliedfluid and substrate voltages and is relatively insensitive to otherelectrodes placed up to 5 mm from the device. This level of control ofthe electric field at the nozzle tip is of significant importance forelectrospray of fluids from a nozzle co-planar with the surface of asubstrate.

[0145] This fine control of the electric field allows for precisecontrol of the electrospray of fluids from these nozzles. Whenelectrospraying fluids from this invention, this fine control of theelectric field allows for a controlled formation of multiple Taylorcones and electrospray plumes from a single nozzle. By simply increasingthe fluid voltage while maintaining the substrate voltage at zero V, thenumber of electrospray plumes emanating from one nozzle can be steppedfrom one to four as illustrated in FIGS. 6 and 7.

[0146] The high electric field at the nozzle tip applies a force to ionscontained within the fluid exiting the nozzle. This force pushespositively-charged ions to the fluid surface when a positive voltage isapplied to the fluid relative to the substrate potential voltage. Due tothe repulsive force of likely-charged ions, the surface area of theTaylor cone generally defines and limits the total number of ions thatcan reside on the fluidic surface. It is generally believed that, forelectrospray, a gas phase ion for an analyte can most easily be formedby that analyte when it resides on the surface of the fluid. The totalsurface area of the fluid increases as the number of Taylor cones at thenozzle tip increases resulting in the increase in solution phase ions atthe surface of the fluid prior to electrospray formation. The ionintensity will increase as measured by the mass spectrometer when thenumber of electrospray plumes increase as shown in the example above.

[0147] Another important feature of the present invention is that sincethe electric field around each nozzle is preferably defined by the fluidand substrate voltage at the nozzle tip, multiple nozzles can be locatedin close proximity, on the order of tens of microns. This novel featureof the present invention allows for the formation of multipleelectrospray plumes from multiple nozzles of a single fluid stream thusgreatly increasing the electrospray sensitivity available formicrochip-based electrospray devices. Multiple nozzles of anelectrospray device in fluid communication with one another not onlyimprove sensitivity but also increase the flow rate capabilities of thedevice. For example, the flow rate of a single fluid stream through onenozzle having the dimensions of a 10 micron inner diameter, 20 micronouter diameter, and a 50 micron length is about 1 μL/min.; and the flowrate through 200 of such nozzles is about 200 μL/min. Accordingly,devices can be fabricated having the capacity for flow rates up to about2 μL/min., from about 2 μL/min. to about 1 mL/min., from about 100nL/min. to about 500 nL/min., and greater than about 2 μL/min. possible.

[0148] Arrays of multiple electrospray devices having any nozzle numberand format may be fabricated according to the present invention. Theelectrospray devices can be positioned to form from a low-density arrayto a high-density array of devices. Arrays can be provided having aspacing between adjacent devices of 9 mm, 4.5 mm, 2.25 mm, 1.12 mm, 0.56mm, 0.28 mm, and smaller to a spacing as close as about 50 μm apart,respectively, which correspond to spacing used in commercialinstrumentation for liquid handling or accepting samples fromelectrospray systems. Similarly, systems of electrospray devices can befabricated in an array having a device density exceeding about 5devices/cm², exceeding about 16 devices/cm², exceeding about 30devices/cm², and exceeding about 81 devices/cm², preferably from about30 devices/cm² to about 100 devices/cm².

[0149] Dimensions of the electrospray device can be determined accordingto various factors such as the specific application, the layout designas well as the upstream and/or downstream device to which theelectrospray device is interfaced or integrated. Further, the dimensionsof the channel and nozzle may be optimized for the desired flow rate ofthe fluid sample. The use of reactive-ion etching techniques allows forthe reproducible and cost effective production of small diameternozzles, for example, a 2 μm inner diameter and 5 μm outer diameter.Such nozzles can be fabricated as close as 20 μm apart, providing adensity of up to about 160,000 nozzles/cm². Nozzle densities up to about10,000/cm², up to about 15,625/cm², up to about 27,566/cm², and up toabout 40,000/cm², respectively, can be provided within an electrospaydevice. Similarly, nozzles can be provided wherein the spacing on theejection surface between the centers of adjacent exit orifices of thespray units is less than about 500 μm, less than about 200 μm, less thanabout 100 μm, and less than about 50 μm, respectively. For example, anelectrospray device having one nozzle with an outer diameter of 20 μmwould respectively have a surrounding sample well 30 μm wide. A denselypacked array of such nozzles could be spaced as close as 50 μm apart asmeasured from the nozzle center.

[0150] In one currently preferred embodiment, the silicon substrate ofthe electrospray device is approximately 250-500 μm in thickness and thecross-sectional area of the through-substrate channel is less thanapproximately 2,500 μm². Where the channel has a circularcross-sectional shape, the channel and the nozzle have an inner diameterof up to 50 μm, more preferably up to 30 μm; the nozzle has an outerdiameter of up to 60 μm, more preferably up to 40 μm; and nozzle has aheight of (and the annular region has a depth of) up to 100 μm. Therecessed portion preferably extends up to 300 μm outwardly from thenozzle. The silicon dioxide layer has a thickness of approximately 1-4μm, preferably 1-3 μm. The silicon nitride layer has a thickness ofapproximately less than 2 μm.

[0151] Furthermore, the electrospray device may be operated to producelarger, minimally-charged droplets. This is accomplished by decreasingthe electric field at the nozzle exit to a value less than that requiredto generate an electrospray of a given fluid. Adjusting the ratio of thepotential voltage of the fluid and the potential voltage of thesubstrate controls the electric field. A fluid to substrate potentialvoltage ratio approximately less than 2 is preferred for dropletformation. The droplet diameter in this mode of operation is controlledby the fluid surface tension, applied voltages and distance to a dropletreceiving well or plate. This mode of operation is ideally suited forconveyance and/or apportionment of a multiplicity of discrete amounts offluids, and may find use in such devices as ink jet printers andequipment and instruments requiring controlled distribution of fluids.

[0152] The electrospray device of the present invention includes asilicon substrate material defining a channel between an entranceorifice on a reservoir surface and a nozzle on a nozzle surface suchthat the electrospray generated by the device is generally perpendicularto the nozzle surface. The nozzle has an inner and an outer diameter andis defined by an annular portion recessed from the surface. The recessedannular region extends radially from the nozzle outer diameter. The tipof the nozzle is co-planar or level with and preferably does not extendbeyond the substrate surface. In this manner the nozzle can be protectedagainst accidental breakage. The nozzle, channel, reservoir and therecessed annular region are etched from the silicon substrate byreactive-ion etching and other standard semiconductor processingtechniques.

[0153] All surfaces of the silicon substrate preferably have insulatinglayers to electrically isolate the liquid sample from the substrate suchthat different potential voltages may be individually applied to thesubstrate and the liquid sample. The insulating layers can constitute asilicon dioxide layer combined with a silicon nitride layer. The siliconnitride layer provides a moisture barrier against water and ions frompenetrating through to the substrate causing electrical breakdownbetween a fluid moving in the channel and the substrate. Theelectrospray apparatus preferably includes at least one controllingelectrode electrically contacting the substrate for the application ofan electric potential to the substrate.

[0154] Preferably, the nozzle, channel and recess are etched from thesilicon substrate by reactive-ion etching and other standardsemiconductor processing techniques. The nozzle side features,through-substrate fluid channel, reservoir side features, andcontrolling electrodes are preferably formed monolithically from amonocrystalline silicon substrate—i.e., they are formed during thecourse of and as a result of a fabrication sequence that requires nomanipulation or assembly of separate components.

[0155] Because the electrospray device is manufactured usingreactive-ion etching and other standard semiconductor processingtechniques, the dimensions of such a device can be very small, forexample, as small as 2 μm inner diameter and 5 μm outer diameter. Thus,a through-substrate fluid channel having, for example, 5 μm innerdiameter and a substrate thickness of 250 μm only has a volume of 4.9pL. The micrometer-scale dimensions of the electrospray device minimizethe dead volume and thereby increase efficiency and analysis sensitivitywhen combined with a separation device.

[0156] The electrospray device of the present invention provides for theefficient and effective formation of an electrospray. By providing anelectrospray surface from which the fluid is ejected with dimensions onthe order of micrometers, the electrospray device limits the voltagerequired to generate a Taylor cone as the voltage is dependent upon thenozzle diameter, the surface tension of the fluid, and the distance ofthe nozzle from an extracting electrode. The nozzle of the electrospraydevice provides the physical asperity on the order of micrometers onwhich a large electric field is concentrated. Further, the electrospraydevice may provide additional electrode(s) on the ejecting surface towhich electric potential(s) may be applied and controlled independent ofthe electric potentials of the fluid and the extracting electrode inorder to advantageously modify and optimize the electric field in orderto focus the gas phase ions resulting from electrospray of fluids. Thecombination of the nozzle and the additional electrode(s) thus enhancethe electric field between the nozzle, the substrate and the extractingelectrode. The electrodes are preferable positioned within about 500microns, and more preferably within about 200 microns from the exitorifice.

[0157] The microchip-based electrospray device of the present inventionprovides minimal extra-column dispersion as a result of a reduction inthe extra-column volume and provides efficient, reproducible, reliableand rugged formation of an electrospray. This electrospray device isperfectly suited as a means of electrospray of fluids frommicrochip-based separation devices. The design of this electrospraydevice is also robust such that the device can be readily mass-producedin a cost-effective, high-yielding process.

[0158] In operation, a conductive or partly conductive liquid sample isintroduced into the through-substrate channel entrance orifice on theinjection surface. The liquid is held at a potential voltage, either bymeans of a conductive fluid delivery device to the electrospray deviceor by means of an electrode formed on the injection surface isolatedfrom the surrounding surface region and from the substrate. The electricfield strength at the tip of the nozzle is enhanced by the applicationof a voltage to the substrate and/or the ejection surface, preferablyzero volts up to approximately less than one-half of the voltage appliedto the fluid. Thus, by the independent control of the fluid/nozzle andsubstrate/ejection surface voltages, the electrospray device of thepresent invention allows the optimization of the electric fieldemanating, from the nozzle. The electrospray device of the presentinvention may be placed 1-2 mm or up to 10 mm from the orifice of anatmospheric pressure ionization (“API”) mass spectrometer to establish astable nanoelectrospray at flow rates in the range of a few nanolitersper minute.

[0159] The electrospray device may be interfaced or integrateddownstream to a sampling device, depending on the particularapplication. For example, the analyte may be electrosprayed onto asurface to coat that surface or into another device for purposes ofconveyance, analysis, and/or synthesis. As described above, highlycharged droplets are formed at atmospheric pressure by the electrospraydevice from nanoliter-scale volumes of an analyte. The highly chargeddroplets produce gas-phase ions upon sufficient evaporation of solventmolecules which may be sampled, for example, through an ion-samplingorifice of an atmospheric pressure ionization mass spectrometer(“API-MS”) for analysis of the electrosprayed fluid.

[0160] One embodiment of the present invention is in the form of anarray of multiple electrospray devices which allows for massive parallelprocessing. The multiple electrospray devices or systems fabricated bymassively parallel processing on a single wafer may then be cut orotherwise separated into multiple devices or systems.

[0161] The electrospray device may also serve to reproducibly distributeand deposit a sample from a mother plate to daughter plate(s) bynanoelectrospray deposition or by the droplet method. A chip-basedcombinatorial chemistry system including a reaction well block maydefine an array of reservoirs for containing the reaction products froma combinatorially synthesized compound. The reaction well block furtherdefines channels, nozzles and recessed portions such that the fluid ineach reservoir may flow through a corresponding channel and exit througha corresponding nozzle in the form of droplets. The reaction well blockmay define any number of reservoir(s) in any desirable configuration,each reservoir being of a suitable dimension and shape. The volume of areservoir may range from a few picoliters up to several microliters.

[0162] The reaction well block may serve as a mother plate to interfaceto a microchip-based chemical synthesis apparatus such that the dropletmethod of the electrospray device may be utilized to reproduciblydistribute discreet quantities of the product solutions to a receivingor daughter plate. The daughter plate defines receiving wells thatcorrespond to each of the reservoirs. The distributed product solutionsin the daughter plate may then be utilized to screen the combinatorialchemical library against biological targets.

[0163] The electrospray device may also serve to reproducibly distributeand deposit an array of samples from a mother plate to daughter plates,for example, for proteomic screening of new drug candidates. This may beby either droplet formation or electrospray modes of operation.Electrospray device(s) may be etched into a microdevice capable ofsynthesizing combinatorial chemical libraries. At a desired time, anozzle(s) may apportion a desired amount of a sample(s) or reagent(s)from a mother plate to a daughter plate(s). Control of the nozzledimensions, applied voltages, and time provide a precise andreproducible method of sample apportionment or deposition from an arrayof nozzles, such as for the generation of sample plates for molecularweight determinations by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (“MALDI-TOFMS”). The capability oftransferring analytes from a mother plate to daughter plates may also beutilized to make other daughter plates for other types of assays, suchas proteomic screening. The fluid to substrate potential voltage ratiocan be chosen for formation of an electrospray or droplet mode based ona particular application.

[0164] An array of multiple electrospray devices can be configured todisperse ink for use in an ink jet printer. The control and enhancementof the electric field at the exit of the nozzles on a substrate willallow for a variation of ink apportionment schemes including theformation of droplets approximately two times the nozzle diameters or ofsubmicometer, highly-charged droplets for blending of different colorsof ink.

[0165] The electrospray device of the present invention can beintegrated with miniaturized liquid sample handling devices forefficient electrospray of the liquid samples for detection using a massspectrometer. The electrospray device may also be used to distribute andapportion fluid samples for use with high-throughput screen technology.The electrospray device may be chip-to-chip or wafer-to-wafer bonded toplastic, glass, or silicon microchip-based liquid separation devicescapable of, for example, capillary electrophoresis, capillaryelectrochromatography, affinity chromatography, liquid chromatography(“LC”), or any other condensed-phase separation technique.

[0166] An array or matrix of multiple electrospray devices of thepresent invention may be manufactured on a single microchip as siliconfabrication using standard, well-controlled thin-film processes. Thisnot only eliminates handling of such micro components but also allowsfor rapid parallel processing of functionally similar elements. The lowcost of these electrospray devices allows for one-time use such thatcross-contamination from different liquid samples may be eliminated.

[0167] FIGS. 13A-13E illustrate the deposition of a discreet sample ontoan electrospray device of the present invention. FIGS. 13A-13C show afluidic probe depositing or transferring a sample to a reservoir on theinjection surface. The fluidic sample is delivered to the reservoir as adiscreet volume generally less than 100 nL. The ‘dots’ representanalytes contained within a fluid. FIG. 13D shows the fluidic samplevolume evaporated leaving the analytes on the reservoir surface. Thisreservoir surface may be coated with a retentive phase, such as ahydrophobic C18-like phase commonly used for LC applications, forincreasing the partition of analytes contained within the fluid to thereservoir surface. FIG. 13E shows a fluidic probe sealed against theinjection surface to deliver a fluidic mobile phase to the microchip toreconstitute the transferred analytes for analysis by electrospray massspectrometry. The probe can have a disposable tip, such as a capillary,micropipette, or microchip.

[0168] A multi-system chip thus provides a rapid sequential chemicalanalysis system fabricated using Micro-ElectroMechanical System (“MEMS”)technology. For example, the multi-system chip enables automated,sequential separation and injection of a multiplicity of samples,resulting in significantly greater analysis throughput and utilizationof the mass spectrometer instrument for, for example, high-throughputdetection of compounds for drug discovery.

[0169] Another aspect of the present invention provides a siliconmicrochip-based electrospray device for producing electrospray of aliquid sample. The electrospray device may be interfaced downstream toan atmospheric pressure ionization mass spectrometer (“API-MS”) foranalysis of the electrosprayed fluid. Another aspect of the invention isan integrated miniaturized liquid phase separation device, which mayhave, for example, glass, plastic or silicon substrates integral withthe electrospray device.

[0170] Electrospray Device Fabrication Procedure

[0171] The electrospray device 250 is preferably fabricated as amonolithic silicon substrate utilizing well-established, controlledthin-film silicon processing techniques such as thermal oxidation,photolithography, reactive-ion etching (RIE), chemical vapor deposition,ion implantation, and metal deposition. Fabrication using such siliconprocessing techniques facilitates massively parallel processing ofsimilar devices, is time- and cost-efficient, allows for tighter controlof critical dimensions, is easily reproducible, and results in a whollyintegral device, thereby eliminating any assembly requirements. Further,the fabrication sequence may be easily extended to create physicalaspects or features on the injection surface and/or ejection surface ofthe electrospray device to facilitate interfacing and connection to afluid delivery system or to facilitate integration with a fluid deliverysub-system to create a single integrated system.

[0172] Nozzle Surface Processing:

[0173] FIGS. 14A-14E and FIGS. 15A-15I illustrate the processing stepsfor the nozzle or ejection side of the substrate in fabricating theelectrospray device of the present invention. Referring to the plan viewof FIG. 14A, a mask is used to pattern 202 that will form the nozzleshape in the completed electrospray device 250. The patterns in the formof circles 204 and 206 forms through-wafer channels and a recessedannular space around the nozzles, respectively of a completedelectrospray device. FIG. 14B is the cross-sectional view taken alongline 14B-14B of FIG. 14A. A double-side polished silicon wafer 200 issubjected to an elevated temperature in an oxidizing environment to growa layer or film of silicon dioxide 210 on the nozzle side and a layer orfilm of silicon dioxide 212 on the reservoir side of the substrate 200.Each of the resulting silicon dioxide layers 210, 212 has a thickness ofapproximately 1-3 μm. The silicon dioxide layers 210, 212 serve as masksfor subsequent selective etching of certain areas of the siliconsubstrate 200.

[0174] A film of positive-working photoresist 208 is deposited on thesilicon dioxide layer 210 on the nozzle side of the substrate 200.Referring to FIG. 14C, an area of the photoresist 204 corresponding tothe entrance to through-wafer channels and an area of photoresistcorresponding to the recessed annular region 206 which will besubsequently etched is selectively exposed through a mask (FIG. 14A) byan optical lithographic exposure tool passing short-wavelength light,such as blue or near-ultraviolet at wavelengths of 365, 405, or 436nanometers.

[0175] As shown in the cross-sectional view of FIG. 14C, afterdevelopment of the photoresist 208, the exposed area 204 of thephotoresist is removed and open to the underlying silicon dioxide layer214 and the exposed area 206 of the photoresist is removed and open tothe underlying silicon dioxide layer 216, while the unexposed areasremain protected by photoresist 208. Referring to FIG. 14D, the exposedareas 214, 216 of the silicon dioxide layer 210 is then etched by afluorine-based plasma with a high degree of anisotropy and selectivityto the protective photoresist 208 until the silicon substrate 218, 220are reached. As shown in the cross-sectional view of FIG. 14E, theremaining photoresist 208 is removed from the silicon substrate 200.

[0176] Referring to the plan view of FIG. 15A, a mask is used to pattern204 in the form of circles. FIG. 15B is the cross-sectional view takenalong line 15B-15B of FIG. 15A. A film of positive-working photoresist208′ is deposited on the silicon dioxide layer 210 on the nozzle side ofthe substrate 200. Referring to FIG. 15C, an area of the photoresist 204corresponding to the entrance to through-wafer channels is selectivelyexposed through a mask (FIG. 15A) by an optical lithographic exposuretool passing short-wavelength light, such as blue or near-ultraviolet atwavelengths of 365, 405, or 436 nanometers.

[0177] As shown in the cross-sectional view of FIG. 15C, afterdevelopment of the photoresist 208′, the exposed area 204 of thephotoresist is removed to the underlying silicon substrate 218. Theremaining photoresist 208′ is used as a mask during the subsequentfluorine based DRIE silicon etch to vertically etch the through-waferchannels 224 shown in FIG. 15D. After etching the through-wafer channels224, the remaining photoresist 208′ is removed from the siliconsubstrate 200.

[0178] As shown in the cross-sectional view of FIG. 15E, the removal ofthe photoresist 208′ exposes the mask pattern of FIG. 14A formed in thesilicon dioxide 210 as shown in FIG. 14E. Referring to FIG. 15F, thesilicon wafer of FIG. 15E is subjected to an elevated temperature in anoxidizing environment to grow a layer or film of silicon dioxide 226,228 on all exposed silicon surfaces of the wafer. Referring to FIG. 15G,the silicon dioxide 226 is then etched by a fluorine-based plasma with ahigh degree of anisotropy and selectivity until the silicon substrate220 is reached. The silicon dioxide layer 228 is designed to serve as anetch stop during the DRIE etch of FIG. 15H that is used to form thenozzle 232 and recessed annular region 230.

[0179] An advantage of the fabrication process described herein is thatthe process simplifies the alignment of the through-wafer channels andthe recessed annular region. This allows the fabrication of smallernozzles with greater ease without any complex alignment of masks.Dimensions of the through channel, such as the aspect ratio (i.e. depthto width), can be reliably and reproducibly limited and controlled.

[0180] Reservoir Surface Processing:

[0181] FIGS. 16A-16I illustrate the processing steps for the reservoiror injection side of the substrate 200 in fabricating the electrospraydevice 250 of the present invention. As shown in the cross-sectionalview in FIG. 16B (a cross-sectional view taken along line 16B-16B ofFIG. 16A), a film of positive-working photoresist 236 is deposited onthe silicon dioxide layer 212. Patterns on the reservoir side arealigned to those previously formed on the nozzle side of the substrateusing through-substrate alignments.

[0182] After alignment, an area of the photoresist 236 corresponding tothe circular reservoir 234 is selectively exposed through a mask (FIG.16A) by an optical lithographic exposure tool passing short-wavelengthlight, such as blue or near-ultraviolet at wavelengths of 365, 405, or436 nanometers. As shown in the cross-sectional view of FIG. 16C, thephotoresist 236 is then developed to remove the exposed areas of thephotoresist 234 such that the reservoir region is open to the underlyingsilicon dioxide layer 238, while the unexposed areas remain protected byphotoresist 236. The exposed area 238 of the silicon dioxide layer 212is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 236 until thesilicon substrate 240 is reached as shown in FIG. 16D.

[0183] As shown in FIG. 16E, a fluorine-based etch creates a cylindricalregion that defines a reservoir 242. The reservoir 242 is etched untilthe through-wafer channels 224 are reached. After the desired depth isachieved the remaining photoresist 236 is then removed in an oxygenplasma or in an actively oxidizing chemical bath like sulfuric acid(H₂SO₄) activated with hydrogen peroxide (H₂O₂), as shown in FIG. 16F.

[0184] Preparation of the Substrate for Electrical Isolation

[0185] Referring to FIG. 16G, the silicon wafer 200 is subjected to anelevated temperature in an oxidizing environment to grow a layer or filmof silicon dioxide 244 on all silicon surfaces to a thickness ofapproximately 1-3 μm. The silicon dioxide layer serves as an electricalinsulating layer. Silicon nitride 246 is further deposited using lowpressure chemical vapor deposition (LPCVD) to provide a conformalcoating of silicon nitride on all surfaces up to 2 μm in thickness, asshown in FIG. 16H. LPCVD silicon nitride also provides furtherelectrical insulation and a fluid barrier that prevents fluids and ionscontained therein that are introduced to the electrospray device fromcausing an electrical connection between the fluid the silicon substrate200. This allows for the independent application of a potential voltageto a fluid and the substrate with this electrospray device to generatethe high electric field at the nozzle tip required for successfulnanoelectrospray of fluids from microchip devices.

[0186] After fabrication of multiple electrospray devices on a singlesilicon wafer, the wafer can be diced or cut into individual devices.This exposes a portion of the silicon substrate 200 as shown in thecross-sectional view of FIG. 16I on which a layer of conductive metal248 is deposited.

[0187] All silicon surfaces are oxidized to form silicon dioxide with athickness that is controllable through choice of temperature and time ofoxidation. All silicon dioxide surfaces are LPCVD coated with siliconnitride. The final thickness of the silicon dioxide and silicon nitridecan be selected to provide the desired degree of electrical isolation inthe device. A thicker layer of silicon dioxide and silicon nitrideprovides a greater resistance to electrical breakdown. The siliconsubstrate is divided into the desired size or array of electrospraydevices for purposes of metalization of the edge of the siliconsubstrate. As shown in FIG. 16I, the edge of the silicon substrate 200is coated with a conductive material 248 using well known thermalevaporation and metal deposition techniques.

[0188] The fabrication method confers superior mechanical stability tothe fabricated electrospray device by etching the features of theelectrospray device from a monocrystalline silicon substrate without anyneed for assembly. The alignment scheme allows for nozzle walls of lessthan 2 μm and nozzle outer diameters down to 5 μm to be fabricatedreproducibly. Further, the lateral extent and shape of the recessedannular region can be controlled independently of its depth. The depthof the recessed annular region also determines the nozzle height and isdetermined by the extent of etch on the nozzle side of the substrate.

[0189] The above described fabrication sequence for the electrospraydevice can be easily adapted to and is applicable for the simultaneousfabrication of a single monolithic system comprising multipleelectrospray devices including multiple channels and/or multipleejection nozzles embodied in a single monolithic substrate. Further, theprocessing steps may be modified to fabricate similar or differentelectrospray devices merely by, for example, modifying the layout designand/or by changing the polarity of the photomask and utilizingnegative-working photoresist rather than utilizing positive-workingphotoresist.

[0190] In a further embodiment an alternate fabrication technique is setforth in FIGS. 17-20. This technique has several advantages over theprior technique, primarily due to the function of the etch stopdeposited on the reservoir side of the substrate. This feature improvesthe production of through-wafer channels having a consistent diameterthroughout its length. An artifact of the etching process is thedifficulty of maintaining consistent channel diameter when approachingan exposed surface of the substrate from within. Typically, the etchingprocess forms a channel having a slightly smaller diameter at the end ofthe channel as it breaks through the opening. This is improved by theability to slightly over-etch the channel when contacting the etch stop.Further, another advantage of etching the reservoir and depositing anetch stop prior to the channel etch is that micro-protrusions resultingfrom the side passivation of the channels remaining at the channelopening are avoided. The etch stop also functions to isolate the plasmaregion from the cooling gas when providing through holes and avoidingpossible contamination from etching by products.

[0191] FIGS. 17A-17E and FIGS. 19A-19G illustrate the processing stepsfor the nozzle or ejection side of the substrate in fabricating theelectrospray device of the present invention. FIGS. 18A-18G illustratethe processing steps for the reservoir or injection side of thesubstrate in fabricating the electrospray device of the presentinvention. FIGS. 20A-20C illustrate the preparation of the substrate forelectrical isolation.

[0192] Referring to the plan view of FIG. 17A, a mask is used to pattern302 that will form the nozzle shape in the completed electrospray device250. The patterns in the form of circles 304 and 306 forms through-waferchannels and a recessed annular space around the nozzles, respectivelyof a completed electrospray device. FIG. 17B is the cross-sectional viewtaken along line 17B-17B of FIG. 17A. A double-side polished siliconwafer 300 is subjected to an elevated temperature in an oxidizingenvironment to grow a layer or film of silicon dioxide 310 on the nozzleside and a layer or film of silicon dioxide 312 on the reservoir side ofthe substrate 300. Each of the resulting silicon dioxide layers 310, 312has a thickness of approximately 1-3 μm. The silicon dioxide layers 310,312 serve as masks for subsequent selective etching of certain areas ofthe silicon substrate 300.

[0193] A film of positive-working photoresist 308 is deposited on thesilicon dioxide layer 310 on the nozzle side of the substrate 300.Referring to FIG. 17C, an area of the photoresist 304 corresponding tothe entrance to through-wafer channels and an area of photoresistcorresponding to the recessed annular region 306 which will besubsequently etched is selectively exposed through a mask (FIG. 17A) byan optical lithographic exposure tool passing short-wavelength light,such as blue or near-ultraviolet at wavelengths of 365, 405, or 436nanometers.

[0194] As shown in the cross-sectional view of FIG. 17C, afterdevelopment of the photoresist 308, the exposed area 304 of thephotoresist is removed and open to the underlying silicon dioxide layer314 and the exposed area 306 of the photoresist is removed and open tothe underlying silicon dioxide layer 310, while the unexposed areasremain protected by photoresist 308. Referring to FIG. 17D, the exposedareas 314, 316 of the silicon dioxide layer 310 is then etched by afluorine-based plasma with a high degree of anisotropy and selectivityto the protective photoresist 308 until the silicon substrate 318, 320are reached. As shown in the cross-sectional view of FIG. 17E, theremaining photoresist 308 is removed from the silicon substrate 300.

[0195] Referring to the plan view of FIG. 18A, a mask is used to pattern324 in the form of a circle. FIG. 18B is the cross-sectional view takenalong line 18B-18B of FIG. 18A. As shown in the cross-sectional view inFIG. 18B a film of positive-working photoresist 326 is deposited on thesilicon dioxide layer 312. Patterns on the reservoir side are aligned tothose previously formed on the nozzle side of the substrate usingthrough-substrate alignments.

[0196] After alignment, an area of the photoresist 326 corresponding tothe circular reservoir 324 is selectively exposed through the mask (FIG.18A) by an optical lithographic exposure tool passing short-wavelengthlight, such as blue or near-ultraviolet at wavelengths of 365, 405, or436 nanometers. As shown in the cross-sectional view of FIG. 18C, thephotoresist 326 is then developed to remove the exposed areas of thephotoresist 324 such that the reservoir region is open to the underlyingsilicon dioxide layer 328, while the unexposed areas remain protected byphotoresist 326. The exposed area 328 of the silicon dioxide layer 312is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 326 until thesilicon substrate 330 is reached as shown in FIG. 18D.

[0197] As shown in FIG. 18E, a fluorine-based etch creates a cylindricalregion that defines a reservoir 332. The reservoir 332 is etched untilthe through-wafer channel depths are reached. After the desired depth isachieved the remaining photoresist 326 is then removed in an oxygenplasma or in an actively oxidizing chemical bath like sulfuric acid(H₂SO₄) activated with hydrogen peroxide (H₂O₂), as shown in FIG. 18F.

[0198] Referring to FIG. 18G, a plasma enhanced chemical vapordeposition (“PECVD”) silicon dioxide layer 334 is deposited on thereservoir side of the substrate 300 to serve as an etch stop for thesubsequent etch of the through substrate channel 336 shown in FIG. 19D.

[0199] A film of positive-working photoresist 308′ is deposited on thesilicon dioxide layer 310 on the nozzle side of the substrate 300, asshown in FIG. 19B. Referring to FIG. 19C, an area of the photoresist 304corresponding to the entrance to through-wafer channels is selectivelyexposed through a mask (FIG. 19A) by an optical lithographic exposuretool passing short-wavelength light, such as blue or near-ultraviolet atwavelengths of 365, 405, or 436 nanometers.

[0200] As shown in the cross-sectional view of FIG. 19C, afterdevelopment of the photoresist 308′, the exposed area 304 of thephotoresist is removed to the underlying silicon substrate 318. Theremaining photoresist 308′ is used as a mask during the subsequentfluorine based DRIE silicon etch to vertically etch the through-waferchannels 336 shown in FIG. 19D. After etching the through-wafer channels336, the remaining photoresist 308′ is removed from the siliconsubstrate 300, as shown in the cross-sectional view of FIG. 19E.

[0201] The removal of the photoresist 308′ exposes the mask pattern ofFIG. 17A formed in the silicon dioxide 310 as shown in FIG. 19E. Thefluorine based DRIE silicon etch is used to vertically etch the recessedannular region 338 shown in FIG. 19F. Referring to FIG. 19G, the silicondioxide layers 310, 312 and 334 are removed from the substrate by ahydrofluoric acid process.

[0202] An advantage of the fabrication process described herein is thatthe process simplifies the alignment of the through-wafer channels andthe recessed annular region. This allows the fabrication of smallernozzles with greater ease without any complex alignment of masks.Dimensions of the through channel, such as the aspect ratio (i.e. depthto width), can be reliably and reproducibly limited and controlled.

[0203] Preparation of the Substrate for Electrical Isolation

[0204] Referring to FIG. 20A, the silicon wafer 300 is subjected to anelevated temperature in an oxidizing environment to grow a layer or filmof silicon dioxide 342 on all silicon surfaces to a thickness ofapproximately 1-3 μm. The silicon dioxide layer serves as an electricalinsulating layer. Silicon nitride 344 is further deposited using lowpressure chemical vapor deposition (LPCVD) to provide a conformalcoating of silicon nitride on all surfaces up to 2 μm in thickness, asshown in FIG. 20B. LPCVD silicon nitride also provides furtherelectrical insulation and a fluid barrier that prevents fluids and ionscontained therein that are introduced to the electrospray device fromcausing an electrical connection between the fluid the silicon substrate300. This allows for the independent application of a potential voltageto a fluid and the substrate with this electrospray device to generatethe high electric field at the nozzle tip required for successfulnanoelectrospray of fluids from microchip devices.

[0205] After fabrication of multiple electrospray devices on a singlesilicon wafer, the wafer can be diced or cut into individual devices.This exposes a portion of the silicon substrate 300 as shown in thecross-sectional view of FIG. 20C on which a layer of conductive metal346 is deposited, which serves as the substrate electrode. A layer ofconductive metal 348 is deposited on the silicon nitride layer of thereservoir side, which serves as the fluid electrode.

[0206] All silicon surfaces are oxidized to form silicon dioxide with athickness that is controllable through choice of temperature and time ofoxidation. All silicon dioxide surfaces are LPCVD coated with siliconnitride. The final thickness of the silicon dioxide and silicon nitridecan be selected to provide the desired degree of electrical isolation inthe device. A thicker layer of silicon dioxide and silicon nitrideprovides a greater resistance to electrical breakdown. The siliconsubstrate is divided into the desired size or array of electrospraydevices for purposes of metalization of the edge of the siliconsubstrate. As shown in FIG. 20C, the edge of the silicon substrate 300is coated with a conductive material 248 using well known thermalevaporation and metal deposition techniques.

[0207] The fabrication methods confer superior mechanical stability tothe fabricated electrospray device by etching the features of theelectrospray device from a monocrystalline silicon substrate without anyneed for assembly. The alignment scheme allows for nozzle walls of lessthan 2 μm and nozzle outer diameters down to 5 μm to be fabricatedreproducibly. Further, the lateral extent and shape of the recessedannular region can be controlled independently of its depth. The depthof the recessed annular region also determines the nozzle height and isdetermined by the extent of etch on the nozzle side of the substrate.

[0208]FIGS. 21A and 21B show a perspective view of scanning electronmicrograph images of a multi-nozzle device fabricated in accordance withthe present invention. The nozzles have a 20 μm outer diameter and an 8μm inner diameter. The pitch, which is the nozzle center to nozzlecenter spacing of the nozzles is 50 μm.

[0209] The above described fabrication sequences for the electrospraydevice can be easily adapted to and are applicable for the simultaneousfabrication of a single monolithic system comprising multipleelectrospray devices including multiple channels and/or multipleejection nozzles embodied in a single monolithic substrate. Further, theprocessing steps may be modified to fabricate similar or differentelectrospray devices merely by, for example, modifying the layout designand/or by changing the polarity of the photomask and utilizingnegative-working photoresist rather than utilizing positive-workingphotoresist.

[0210] Interface of a Multi-System Chip to a Mass Spectrometer

[0211] Arrays of electrospray nozzles on a multi-system chip may beinterfaced with a sampling orifice of a mass spectrometer by positioningthe nozzles near the sampling orifice. The tight configuration ofelectrospray nozzles allows the positioning thereof in close proximityto the sampling orifice of a mass spectrometer.

[0212] A multi-system chip may be manipulated relative to the ionsampling orifice to position one or more of the nozzles for electrospraynear the sampling orifice. Appropriate voltage(s) may then be applied tothe one or more of the nozzles for electrospray.

[0213] Although the invention has been described in detail for thepurpose of illustration, it is understood that such detail is solely forthat purpose, and variations can be made therein by those skilled in theart without departing from the spirit and scope of the invention whichis defined by the following claims.

What is claimed is:
 1. An electrospray device for generating multiplesprays from a single fluid stream comprising: a substrate having: a) aninjection surface; b) an ejection surface opposing the injectionsurface, wherein the substrate is an integral monolith having either i)a plurality of spray units each capable of generating a singleelectrospray plume wherein the entrance orifice of each spray unit is influid communication with one another or ii) a plurality of spray unitseach capable of generating multiple electrospray plumes wherein theentrance orifice of each spray unit is in fluid communication with oneanother or iii) a single spray unit capable of generating multipleelectrospray plumes, for spraying the fluid, each spray unit comprising:an entrance orifice on the injection surface, an exit orifice on theejection surface, a channel extending between the entrance orifice andthe exit orifice, and a recess surrounding the exit orifice positionedbetween the injection surface and the ejection surface; and c) anelectric field generating source positioned to define an electric fieldsurrounding at least one exit orifice.
 2. The electrospray deviceaccording to claim 1, wherein the substrate has a plurality of sprayunits each capable of generating a single electrospray plume wherein theentrance orifice of each spray unit is in fluid communication with oneanother.
 3. The electrospray device according to claim 1, wherein thesubstrate has a plurality of spray units each capable of generatingmultiple electrospray plumes wherein the entrance orifice of each sprayunit is in fluid communication with one another.
 4. The electrospraydevice according to claim 1, wherein the substrate has a single sprayunit capable of generating multiple electrospray plumes.
 5. Theelectrospray device according to claim 2, wherein the plurality of sprayunits are configured to generate a single combined electrospray plume offluid.
 6. The electrospray device according to claim 3, wherein at leastone of the spray units is configured to generate multiple electrosprayplumes of fluid which remain discrete.
 7. The electrospray deviceaccording to claim 3, wherein the plurality of spray units areconfigured to generate a single combined electrospray plume of fluid. 8.The electrospray device according to claim 4, wherein the single sprayunit is configured to generate multiple electrospray plumes of fluidwhich remain discrete.
 9. The electrospray device of claim 2, whereinthe exit orifices of the spray units are present on the ejection surfaceat a density of up to about 10,000 exit orifices/cm².
 10. Theelectrospray device of claim 2, wherein the exit orifices of the sprayunits are present on the ejection surface at a density of up to about15,625 exit orifices/cm².
 11. The electrospray device of claim 2,wherein the exit orifices of the spray units are present on the ejectionsurface at a density of up to about 27,566 exit orifices/cm².
 12. Theelectrospray device of claim 2, wherein the exit orifices of the sprayunits are present on the ejection surface at a density of up to about40,000 exit orifices/cm².
 13. The electrospray device of claim 2,wherein the exit orifices of the spray units are present on the ejectionsurface at a density of up to about 160,000 exit orifices/cm².
 14. Theelectrospray device of claim 3, wherein the exit orifices of the sprayunits are present on the ejection surface at a density of up to about10,000 exit orifices/cm².
 15. The electrospray device of claim 3,wherein the exit orifices of the spray units are present on the ejectionsurface at a density of up to about 15,625 exit orifices/cm².
 16. Theelectrospray device of claim 3, wherein the exit orifices of the sprayunits are present on the ejection surface at a density of up to about27,566 exit orifices/cm².
 17. The electrospray device of claim 3,wherein the exit orifices of the spray units are present on the ejectionsurface at a density of up to about 40,000 exit orifices/cm².
 18. Theelectrospray device of claim 3, wherein the exit orifices of the sprayunits are present on the ejection surface at a density of up to about160,000 exit orifices/cm².
 19. The electrospray device of claim 2,wherein the spacing on the ejection surface between the centers ofadjacent exit orifices of the spray units is less than about 500 μm. 20.The electrospray device of claim 2, wherein the spacing on the ejectionsurface between the centers of adjacent exit orifices of the spray unitsis less than about 200 μm.
 21. The electrospray device of claim 2,wherein the spacing on the ejection surface between the centers ofadjacent exit orifices of the spray units is less than about 100 μm. 22.The electrospray device of claim 2, wherein the spacing on the ejectionsurface between the centers of adjacent exit orifices of the spray unitsis less than about 50 μm.
 23. The electrospray device of claim 3,wherein the spacing on the ejection surface between the centers ofadjacent exit orifices of the spray units is less than about 500 μm. 24.The electrospray device of claim 3, wherein the spacing on the ejectionsurface between the centers of adjacent exit orifices of the spray unitsis less than about 200 μm.
 25. The electrospray device of claim 3,wherein the spacing on the ejection surface between the centers ofadjacent exit orifices of the spray units is less than about 100 μm. 26.The electrospray device of claim 3, wherein the spacing on the ejectionsurface between the centers of adjacent exit orifices of the spray unitsis less than about 50 μm.
 27. The electrospray device according to claim1, wherein said substrate comprises silicon.
 28. The electrospray deviceaccording to claim 1, wherein said substrate is polymeric.
 29. Theelectrospray device according to claim 1, wherein said substratecomprises glass.
 30. The electrospray device according to claim 2,wherein said electric field generating source comprises: a firstelectrode attached to said substrate to impart a first potential to saidsubstrate; and a second electrode to impart a second potential, whereinthe first and the second electrodes are positioned to define an electricfield surrounding at least one exit orifice.
 31. The electrospray deviceaccording to claim 30, wherein the first electrode is electricallyinsulated from the fluid and the second potential is applied to thefluid.
 32. The electrospray device according to claim 30, wherein thefirst electrode is in electrical contact with the fluid and the secondelectrode is positioned on the ejection surface.
 33. The electrospraydevice according to claim 30, wherein application of potentials to saidfirst and second electrodes causes the fluid to discharge from at leastone exit orifice in the form of an electrospray plume.
 34. Theelectrospray device according to claim 3, wherein said electric fieldgenerating source comprises: a first electrode attached to saidsubstrate to impart a first potential to said substrate; and a secondelectrode to impart a second potential, wherein the first and the secondelectrodes are positioned to define an electric field surrounding atleast one exit orifice.
 35. The electrospray device according to claim34, wherein the first electrode is electrically insulated from the fluidand the second potential is applied to the fluid.
 36. The electrospraydevice according to claim 34, wherein the first electrode is inelectrical contact with the fluid and the second electrode is positionedon the ejection surface.
 37. The electrospray device according to claim34, wherein application of potentials to said first and secondelectrodes causes the fluid to discharge from at least one exit orificein the form of multiple electrospray plumes.
 38. The electrospray deviceaccording to claim 4, wherein said electric field generating sourcecomprises: a first electrode attached to said substrate to impart afirst potential to said substrate; and a second electrode to impart asecond potential, wherein the first and the second electrodes arepositioned to define an electric field surrounding the exit orifice. 39.The electrospray device according to claim 38, wherein the firstelectrode is electrically insulated from the fluid and the secondpotential is applied to the fluid.
 40. The electrospray device accordingto claim 38, wherein the first electrode is in electrical contact withthe fluid and the second electrode is positioned on the ejectionsurface.
 41. The electrospray device according to claim 38, whereinapplication of potentials to said first and second electrodes causes thefluid to discharge from the orifice in the form of multiple electrosprayplumes.
 42. The electrospray device according to claim 30, wherein saidfirst electrode is positioned within 500 microns of the exit orifice.43. The electrospray device according to claim 30, wherein said firstelectrode is positioned within 200 microns of the exit orifice.
 44. Theelectrospray device according to claim 30, wherein said second electrodeis positioned within 500 microns of the exit orifice.
 45. Theelectrospray device according to claim 30, wherein said second electrodeis positioned within 200 microns of the exit orifice.
 46. Theelectrospray device according to claim 30, wherein the exit orifice hasa distal end in conductive contact with the substrate.
 47. Theelectrospray device according to claim 34, wherein said first electrodeis positioned within 500 microns of the exit orifice.
 48. Theelectrospray device according to claim 34, wherein said first electrodeis positioned within 200 microns of the exit orifice.
 49. Theelectrospray device according to claim 34, wherein said second electrodeis positioned within 500 microns of the exit orifice.
 50. Theelectrospray device according to claim 34, wherein said second electrodeis positioned within 200 microns of the exit orifice.
 51. Theelectrospray device according to claim 34, wherein the exit orifice hasa distal end in conductive contact with the substrate.
 52. Theelectrospray device according to claim 38, wherein said first electrodeis positioned within 500 microns of the exit orifice.
 53. Theelectrospray device according to claim 38, wherein said first electrodeis positioned within 200 microns of the exit orifice.
 54. Theelectrospray device according to claim 38, wherein said second electrodeis positioned within 500 microns of the exit orifice.
 55. Theelectrospray device according to claim 38, wherein said second electrodeis positioned within 200 microns of the exit orifice.
 56. Theelectrospray device according to claim 38, wherein the exit orifice hasa distal end in conductive contact with the substrate.
 57. Theelectrospray device according to claim 4, wherein the device isconfigured to permit an electrospray of fluid at a flow rate of up toabout 2 μL/minute.
 58. The electrospray device according to claim 4,wherein the device is configured to permit an electrospray of fluid at aflow rate of from about 100 nL/minute to about 500 nL/minute.
 59. Theelectrospray device according to claim 2, wherein the device isconfigured to permit an electrospray of fluid at a flow rate of up toabout 2 μL/minute.
 60. The electrospray device according to claim 2,wherein the device is configured to permit an electrospray of fluid at aflow rate of greater than about 2 μL/minute.
 61. The electrospray deviceaccording to claim 60, wherein the flow rate is from about 2 μL/minuteto about 1 mL/minute.
 62. The electrospray device according to claim 60,wherein the flow rate is from about 100 nL/minute to about 500nL/minute.
 63. The electrospray device according to claim 3, wherein thedevice is configured to permit an electrospray of fluid at a flow rateof up to about 2 μL/minute.
 64. The electrospray device according toclaim 3, wherein the device is configured to permit an electrospray offluid at a flow rate of greater than about 2 μL/minute.
 65. Theelectrospray device according to claim 64, wherein the flow rate is fromabout 2 μL/minute to about 1 mL/minute.
 66. The electrospray deviceaccording to claim 64, wherein the flow rate is from about 100 nL/minuteto about 500 nL/minute.
 67. An electrospray system for spraying fluidcomprising an array of a plurality of electrospray devices of claim 1.68. The electrospray system according to claim 67, wherein theelectrospray device density in the array exceeds about 5 devices/cm².69. The electrospray system according to claim 67, wherein theelectrospray device density in the array exceeds about 16 devices/cm².70. The electrospray system according to claim 67, wherein theelectrospray device density in the array exceeds about 30 devices/cm².71. The electrospray system according to claim 67, wherein theelectrospray device density in the array exceeds about 81 devices/cm².72. The electrospray system according to claim 67, wherein theelectrospray device density in the array is from about 30 devices/cm² toabout 100 devices/cm².
 73. The electrospray system according to claim67, wherein said array is an integral monolith of said devices.
 74. Theelectrospray system according to claim 67, wherein at least two of thedevices are in fluid communication with different fluid streams.
 75. Theelectrospray system according to claim 67, wherein at least one sprayunit is configured to generate multiple electrospray plumes of fluid.76. The electrospray system according to claim 67, wherein at least oneof the electrospray devices is configured to generate a single combinedelectrospray plume of fluid.
 77. The electrospray system according toclaim 67, wherein at least one spray unit of the plurality of sprayunits is configured to generate a single electrospray plume of fluid.78. The electrospray system according to claim 67, wherein at least onespray unit of the plurality of spray units is configured to generatemultiple electrospray plumes of fluid which remain discrete.
 79. Theelectrospray system according to claim 67, wherein said substratecomprises silicon.
 80. The electrospray system according to claim 67,wherein said substrate is polymeric.
 81. The electrospray systemaccording to claim 67, wherein said substrate comprises glass.
 82. Theelectrospray system according to claim 67, wherein at least one devicecomprises a substrate having a plurality of spray units each capable ofgenerating a single electrospray plume wherein the entrance orifice ofeach spray unit is in fluid communication with one another.
 83. Theelectrospray system according to claim 67, wherein at least one devicecomprises a substrate having a plurality of spray units each capable ofgenerating multiple electrospray plumes wherein the entrance orifice ofeach spray unit is in fluid communication with one another.
 84. Theelectrospray system according to claim 67, wherein at least one devicecomprises a substrate having a single spray unit capable of generatingmultiple electrospray plumes.
 85. The electrospray system according toclaim 82, wherein the plurality of spray units are configured togenerate a single combined electrospray plume of fluid.
 86. Theelectrospray system according to claim 83, wherein at least one of thespray units is configured to generate multiple electrospray plumes offluid which remain discrete.
 87. The electrospray system according toclaim 83, wherein the plurality of spray units are configured togenerate a single combined electrospray plume of fluid.
 88. Theelectrospray system according to claim 84, wherein the single spray unitis configured to generate multiple electrospray plumes of fluid whichremain discrete.
 89. The electrospray system of claim 82, wherein in atleast one device the exit orifices of the spray units are present on theejection surface at a density of up to about 10,000 exit orifices/cm².90. The electrospray system of claim 82, wherein in at least one devicethe exit orifices of the spray units are present on the ejection surfaceat a density of up to about 15,625 exit orifices/cm².
 91. Theelectrospray system of claim 82, wherein in at least one device the exitorifices of the spray units are present on the ejection surface at adensity of up to about 27,566 exit orifices/cm².
 92. The electrospraysystem of claim 82, wherein in at least one device the exit orifices ofthe spray units are present on the ejection surface at a density of upto about 40,000 exit orifices/cm².
 93. The electrospray system of claim82, wherein in at least one device the exit orifices of the spray unitsare present on the ejection surface at a density of up to about 160,000exit orifices/cm².
 94. The electrospray system of claim 83, wherein inat least one device the exit orifices of the spray units are present onthe ejection surface at a density of up to about 10,000 exitorifices/cm².
 95. The electrospray system of claim 83, wherein in atleast one device the exit orifices of the spray units are present on theejection surface at a density of up to about 15,625 exit orifices/cm².96. The electrospray system of claim 83, wherein in at least one devicethe exit orifices of the spray units are present on the ejection surfaceat a density of up to about 27,566 exit orifices/cm².
 97. Theelectrospray system of claim 83, wherein in at least one device the exitorifices of the spray units are present on the ejection surface at adensity of up to about 40,000 exit orifices/cm².
 98. The electrospraysystem of claim 83, wherein in at least one device the exit orifices ofthe spray units are present on the ejection surface at a density of upto about 160,000 exit orifices/cm².
 99. The electrospray system of claim82, wherein in at least one device the spacing on the ejection surfacebetween the centers of adjacent exit orifices of the spray units is lessthan about 500 μm.
 100. The electrospray system of claim 83, wherein inat least one device the spacing on the ejection surface between thecenters of adjacent exit orifices of the spray units is less than about200 μm.
 101. The electrospray system of claim 83, wherein in at leastone device the spacing on the ejection surface between the centers ofadjacent exit orifices of the spray units is less than about 100 μm.102. The electrospray system of claim 82, wherein in at least one devicethe spacing on the ejection surface between the centers of adjacent exitorifices of the spray units is less than about 50 μm.
 103. Theelectrospray system of claim 83, wherein in at least one device thespacing on the ejection surface between the centers of adjacent exitorifices of the spray units is less than about 500 μm.
 104. Theelectrospray system of claim 83, wherein in at least one device thespacing on the ejection surface between the centers of adjacent exitorifices of the spray units is less than about 200 μm.
 105. Theelectrospray system of claim 83, wherein in at least one device thespacing on the ejection surface between the centers of adjacent exitorifices of the spray units is less than about 100 μm.
 106. Theelectrospray system of claim 83, wherein in at least one device thespacing on the ejection surface between the centers of adjacent exitorifices of the spray units is less than about 50 μm.
 107. Theelectrospray system according to claim 82, wherein said electric fieldgenerating source comprises: a first electrode attached to saidsubstrate to impart a first potential to said substrate; and a secondelectrode to impart a second potential, wherein the first and the secondelectrodes are positioned to define an electric field surrounding atleast one exit orifice.
 108. The electrospray system according to claim107, wherein the first electrode is electrically insulated from thefluid and the second potential is applied to the fluid.
 109. Theelectrospray system according to claim 107, wherein the first electrodeis in electrical contact with the fluid and the second electrode ispositioned on the ejection surface.
 110. The electrospray systemaccording to claim 107, wherein application of potentials to said firstand second electrodes causes the fluid to discharge from at least oneexit orifice in the form of an electrospray plume.
 111. The electrospraysystem according to claim 83, wherein said electric field generatingsource comprises: a first electrode attached to said substrate to imparta first potential to said substrate; and a second electrode to impart asecond potential, wherein the first and the second electrodes arepositioned to define an electric field surrounding at least one exitorifice.
 112. The electrospray system according to claim 111, whereinthe first electrode is electrically insulated from the fluid and thesecond potential is applied to the fluid.
 113. The electrospray systemaccording to claim 111, wherein the first electrode is in electricalcontact with the fluid and the second electrode is positioned on theejection surface.
 114. The electrospray system according to claim 111,wherein application of potentials to said first and second electrodescauses the fluid to discharge from at least one exit orifice in the formof multiple electrospray plumes.
 115. The electrospray system accordingto claim 84, wherein said electric field generating source comprises: afirst electrode attached to said substrate to impart a first potentialto said substrate; and a second electrode to impart a second potential,wherein the first and the second electrodes are positioned to define anelectric field surrounding the exit orifice.
 116. The electrospraysystem according to claim 115, wherein the first electrode iselectrically insulated from the fluid and the second potential isapplied to the fluid.
 117. The electrospray system according to claim115, wherein the first electrode is in electrical contact with the fluidand the second electrode is positioned on the ejection surface.
 118. Theelectrospray system according to claim 115, wherein application ofpotentials to said first and second electrodes causes the fluid todischarge from the orifice in the form of multiple electrospray plumes.119. The electrospray system according to claim 107, wherein said firstelectrode is positioned within 200 microns of the exit orifice.
 120. Theelectrospray system according to claim 107, wherein said secondelectrode is positioned within 200 microns of the exit orifice.
 121. Theelectrospray system according to claim 107, wherein the exit orifice hasa distal end in conductive contact with the substrate.
 122. Theelectrospray system according to claim 111, wherein said first electrodeis positioned within 200 microns of the exit orifice.
 123. Theelectrospray system according to claim 111, wherein said secondelectrode is positioned within 200 microns of the exit orifice.
 124. Theelectrospray system according to claim 111, wherein the exit orifice hasa distal end in conductive contact with the substrate.
 125. Theelectrospray system according to claim 115, wherein said first electrodeis positioned within 200 microns of the exit orifice.
 126. Theelectrospray system according to claim 115, wherein said secondelectrode is positioned within 200 microns of the exit orifice.
 127. Theelectrospray system according to claim 115, wherein the exit orifice hasa distal end in conductive contact with the substrate.
 128. Theelectrospray system according to claim 84, wherein at least one deviceis configured to permit an electrospray of fluid at a flow rate of up toabout 2 μL/minute.
 129. The electrospray system according to claim 84,wherein at least one device is configured to permit an electrospray offluid at a flow rate of from about 100 nL/minute to about 500 nL/minute.130. The electrospray system according to claim 82, wherein the deviceis configured to permit an electrospray of fluid at a flow rate of up toabout 2 μL/minute.
 131. The electrospray system according to claim 82,wherein the device is configured to permit an electrospray of fluid at aflow rate of greater than about 2 μL/minute.
 132. The electrospraysystem according to claim 131, wherein the flow rate is from about 2μL/minute to about 1 mL/minute.
 133. The electrospray system accordingto claim 131, wherein the flow rate is from about 100 nL/minute to about500 nL/minute.
 134. The electrospray system according to claim 83,wherein at least one device is configured to permit an electrospray offluid at a flow rate of up to about 2 μL/minute.
 135. The electrospraysystem according to claim 83, wherein at least one device is configuredto permit an electrospray of fluid at a flow rate of greater than about2 μL/minute.
 136. The electrospray system according to claim 135,wherein the flow rate is from about 2 μL/minute to about 1 mL/minute.137. The electrospray system according to claim 135, wherein the flowrate is from about 100 nL/minute to about 500 nL/minute.
 138. Theelectrospray system according to claim 67, wherein the spacing on theejection surface between adjacent devices is about 9 mm or less. 139.The electrospray system according to claim 67, wherein the spacing onthe ejection surface between adjacent devices is about 4.5 mm or less.140. The electrospray system according to claim 67, wherein the spacingon the ejection surface between adjacent devices is about 2.2 mm orless.
 141. The electrospray system according to claim 67, wherein thespacing on the ejection surface between adjacent devices is about 1.1 mmor less.
 142. The electrospray system according to claim 67, wherein thespacing on the ejection surface between adjacent devices is about 0.56mm or less.
 143. The electrospray system according to claim 67, whereinthe spacing on the ejection surface between adjacent devices is about0.28 mm or less.
 144. The electrospray system according to claim 82,wherein the spacing on the ejection surface between adjacent devices isabout 9 mm or less.
 145. The electrospray system according to claim 82,wherein the spacing on the ejection surface between adjacent devices isabout 4.5 mm or less.
 146. The electrospray system according to claim82, wherein the spacing on the ejection surface between adjacent devicesis about 2.2 mm or less.
 147. The electrospray system according to claim82, wherein the spacing on the ejection surface between adjacent devicesis about 1.1 mm or less.
 148. The electrospray system according to claim82, wherein the spacing on the ejection surface between adjacent devicesis about 0.56 mm or less.
 149. The electrospray system according toclaim 82, wherein the spacing on the ejection surface between adjacentdevices is about 0.28 mm or less.
 150. The electrospray system accordingto claim 83, wherein the spacing on the ejection surface betweenadjacent devices is about 9 mm or less.
 151. The electrospray systemaccording to claim 83, wherein the spacing on the ejection surfacebetween adjacent devices is about 4.5 mm or less.
 152. The electrospraysystem according to claim 83, wherein the spacing on the ejectionsurface between adjacent devices is about 2.2 mm or less.
 153. Theelectrospray system according to claim 83, wherein the spacing on theejection surface between adjacent devices is about 1.1 mm or less. 154.The electrospray system according to claim 83, wherein the spacing onthe ejection surface between adjacent devices is about 0.56 mm or less.155. The electrospray system according to claim 83, wherein the spacingon the ejection surface between adjacent devices is about 0.28 mm orless.
 156. The electrospray system according to claim 84, wherein thespacing on the ejection surface between adjacent devices is about 9 mmor less.
 157. The electrospray system according to claim 84, wherein thespacing on the ejection surface between adjacent devices is about 4.5 mmor less.
 158. The electrospray system according to claim 84, wherein thespacing on the ejection surface between adjacent devices is about 2.2 mmor less.
 159. The electrospray system according to claim 84, wherein thespacing on the ejection surface between adjacent devices is about 1.1 mmor less.
 160. The electrospray system according to claim 84, wherein thespacing on the ejection surface between adjacent devices is about 0.56mm or less.
 161. The electrospray system according to claim 84, whereinthe spacing on the ejection surface between adjacent devices is about0.28 mm or less.
 162. A system for processing multiple sprays of fluidcomprising: an electrospray device according to claim 1 and a device toreceive multiple sprays of fluid from said electrospray device.
 163. Thesystem according to claim 162, wherein the device to receive multiplesprays of fluid receives electrospray plumes of the fluid emanating froma plurality of the spray units of said electrospray device.
 164. Thesystem according to claim 163, wherein multiple electrospray plumes ofthe fluid emanate from at least one of the plurality of spray units ofsaid electrospray device.
 165. The system according to claim 162,wherein the device to receive multiple sprays of fluid receives multipleelectrospray plumes of the fluid emanating from the single spray unit ofsaid electrospray device.
 166. The system according to claim 162,wherein the device to receive multiple sprays of fluid receives dropletsof the fluid emanating from a plurality of spray units of saidelectrospray device.
 167. The system according to claim 162, whereinsaid device to receive multiple sprays of fluid comprises a surface forreceiving said fluid.
 168. The system according to claim 167, whereinsaid surface comprises a daughter plate or MALDI sample plate, having aplurality of fluid receiving wells each positioned to receive fluidejected from said electrospray device.
 169. The system according toclaim 162, wherein said device to receive multiple sprays of fluid is amass spectrometry device.
 170. A system for processing multiple spraysof fluid comprising: an electrospray system according to claim 67 and adevice to receive multiple sprays of fluid from said electrospraysystem.
 171. The system according to claim 170, wherein the device toreceive multiple sprays of fluid receives electrospray plumes of thefluid emanating from a plurality of the spray units of said electrospraysystem.
 172. The system according to claim 171, wherein multipleelectrospray plumes of the fluid emanate from at least one of the sprayunits of said electrospray system.
 173. The system according to claim170, wherein the device to receive multiple sprays of fluid receivesdroplets of the fluid emanating from a plurality of spray units of saidelectrospray system.
 174. The system according to claim 170, whereinsaid device to receive multiple sprays of fluid comprises a surface forreceiving said fluid.
 175. The system according to claim 174, whereinsaid surface comprises: a daughter plate or MALDI sample plate, having aplurality of fluid receiving wells each positioned to receive fluidejected from said electrospray system.
 176. The system according toclaim 170, wherein said device to receive multiple sprays of fluid is amass spectrometry device.
 177. A system for processing multiple spraysof fluid comprising: an electrospray device according to claim 1 and adevice to provide at least one sample in solution or fluid orcombination thereof to at least one entrance orifice of saidelectrospray device.
 178. The system according to claim 177, wherein atleast one of: a) the entrance orifices of the plurality of spray unitsof said electrospray device are in fluid communication with one anotherby a first reservoir, and b) the entrance orifice of the single sprayunit is in fluid communication with a second reservoir; and wherein saiddevice to provide at least one sample in solution or fluid orcombination thereof to at least one entrance orifice comprises: at leastone conduit to provide delivery of at least one sample in solution orfluid or combination thereof to at least one reservoir of said device.179. The system according to claim 177, wherein said at least oneconduit comprises a capillary, micropipette, or microchip.
 180. Thesystem according to claim 177, wherein the at least one conduit andreservoir provide a fluid tight seal therebetween, said at least oneconduit optionally comprising a disposable tip.
 181. The systemaccording to claim 177, wherein said at least one conduit is compatiblewith mutiple entrance orifices and is repositionable from one entranceorifice to another entrance orifice.
 182. The system according to claim181, wherein said at least one conduit is capable of being receded fromone entrance orifice and repositioned in line with another entranceorifice and placed in sealing engagement with the another entranceorifice to provide fluid thereto.
 183. The system according to claim177, wherein said device to provide at least one sample in solution orfluid or combination thereof to at least one entrance orifice of saidelectrospray device carries out liquid separation analysis on the fluid.184. The system according to claim 183, wherein the liquid separationanalysis is capillary electrophoresis, capillary dielectrophoresis,capillary electrochromatography, or liquid chromatography.
 185. A systemfor processing multiple sprays of fluid comprising: a system accordingto claim 177 and a device to receive multiple sprays of fluid from saidelectrospray device.
 186. The system according to claim 185, wherein thedevice to receive multiple sprays of fluid receives plumes of the fluidemanating from a plurality of the spray units of said electrospraydevice.
 187. The system according to claim 185, wherein the device toreceive multiple sprays of fluid receives multiple electrospray plumesof the fluid emanating from at least one spray unit of said electrospraydevice
 188. The system according to claim 185, wherein said device toreceive multiple sprays of fluid comprises a surface for receiving saidfluid.
 189. The system according to claim 188, wherein said surfacecomprises: a daughter plate or MALDI sample plate, having a plurality offluid receiving wells each positioned to receive fluid ejected from saidelectrospray system.
 190. The system according to claim 185, whereinsaid device to receive multiple sprays of fluid is a mass spectrometrydevice.
 191. A system for processing multiple sprays of fluidcomprising: an electrospray system according to claim 67 and a device toprovide at least one sample in solution or fluid or combination thereofto at least one entrance orifice of said electrospray system.
 192. Thesystem according to claim 191, wherein at least one of: a) the entranceorifices of the plurality of spray units of said electrospray device arein fluid communication with one another by a first reservoir, and b) theentrance orifice of the single spray unit is in fluid communication witha second reservoir; and wherein said device to provide at least onesample in solution or fluid or combination thereof to at least oneentrance orifice comprises: at least one conduit to provide delivery ofat least one sample in solution or fluid or combination thereof to atleast one reservoir of said device.
 193. The system according to claim191, wherein said at least one conduit comprises a capillary,micropipette, or microchip.
 194. The system according to claim 191,wherein the at least one conduit and reservoir provide a fluid tightseal therebetween, said at least one conduit optionally comprising adisposable tip.
 195. The system according to claim 191, wherein said atleast one conduit is compatible with multiple entrance orifices and isrepositionable from one entrance orifice to another entrance orifice.196. The system according to claim 195, wherein said at least oneconduit is capable of being receded from one entrance orifice andrepositioned in line with another entrance orifice and placed in sealingengagement with the another entrance orifice to provide fluid thereto.197. The system according to claim 191, wherein said device to provideat least one sample in solution or fluid or combination thereof to atleast one entrance orifice of said electrospray device carries outliquid separation analysis on the fluid.
 198. The system according toclaim 197, wherein the liquid separation analysis is capillaryelectrophoresis, capillary dielectrophoresis, capillaryelectrochromatography, or liquid chromatography.
 199. A system forprocessing multiple sprays of fluid comprising: a system according toclaim 191 and a device to receive multiple sprays of fluid from saidelectrospray system.
 200. The system according to claim 199, wherein thedevice to receive multiple sprays of fluid receives plumes of the fluidemanating from a plurality of the spray units of said electrospraysystem.
 201. The system according to claim 199, wherein the device toreceive multiple sprays of fluid receives multiple electrospray plumesof the fluid emanating from at least one spray unit of said electrospraysystem.
 202. The system according to claim 199, wherein said device toreceive multiple sprays of fluid comprises a surface for receiving saidfluid.
 203. The system according to claim 202, wherein said surfacecomprises: a daughter plate or MALDI sample plate, having a plurality offluid receiving wells each positioned to receive fluid ejected from saidelectrospray system.
 204. The system according to claim 199, whereinsaid device to receive multiple sprays of fluid is a mass spectrometrydevice.
 205. A method for processing multiple sprays of fluidcomprising: providing an electrospray device according to claim 1;providing a device to provide at least one fluid sample to at least oneentrance orifice of said electrospray device; providing a device toreceive multiple sprays of fluid or droplets from said electrospraydevice; passing a fluid from said fluid providing device to saidelectrospray device; generating an electric filed surrounding the exitorifice of said at least one spray unit such that fluid dischargedtherefrom forms an electrospray or droplets; and passing saidelectrospray or droplets from said electrospray device to said receivingdevice.
 206. The method of claim 205, further comprising using saidreceiving device for performing mass spectrometry analysis, liquidchromatography analysis, or protein, DNA, or RNA combinatorial chemistryanalysis.
 207. A method for processing multiple sprays of fluidcomprising: providing an electrospray system according to claim 67;providing a device to provide at least one fluid sample to at least oneentrance orifice of at least one electrospray device of saidelectrospray system; providing a device to receive multiple sprays offluid or droplets from said at least one electrospray device; passing afluid from said fluid providing device to said at least one electrospraydevice; generating an electric filed surrounding an exit orifice of atleast one spray unit within said at least one electrospray device suchthat fluid discharged therefrom forms an electrospray or droplets; andpassing said electrospray or droplets from said at least oneelectrospray device to said receiving device.
 208. The method of claim207, further comprising using said receiving device for performing massspectrometry analysis, liquid chromatography analysis, or protein, DNA,or RNA combinatorial chemistry analysis.
 209. A method of generating anelectrospray comprising: providing an electrospray device according toclaim 1; passing a fluid into the entrance orifice, through the channel,and through the exit orifice of at least one spray unit; generating anelectric field surrounding the exit orifice of said at least one sprayunit such that fluid discharged therefrom forms an electrospray. 210.The method according to claim 209, further comprising: detectingcomponents of the electrospray by spectroscopic detection.
 211. Themethod according to claim 210, wherein the spectroscopic detection isselected from the group consisting of UV absorbance, laser inducedfluorescence, and evaporative light scattering.
 212. The methodaccording to claim 209, wherein the fluid is discharged at a flow rateof up to about 2 μL/minute.
 213. The method according to claim 209,wherein the fluid is discharged at a flow rate of greater than about 2μL/minute.
 214. The method according to claim 209, wherein the fluid isdischarged at a flow rate of from about 2 μL/minute to about 1μmL/minute.
 215. The method according to claim 209, wherein the fluid isdischarged at a flow rate of from about 100 nL/minute to about 500nL/minute.
 216. A method of mass spectrometric analysis comprising:providing the system according to claim 162, wherein the device toreceive multiple sprays of fluid from said electrospray device is a massspectrometer; passing a fluid into the entrance orifice, through thechannel, and through the exit orifice of at least one spray unit underconditions effective to produce an electrospray; and passing theelectrospray into the mass spectrometer, whereby the fluid is subjectedto a mass spectrometry analysis.
 217. The method according to claim 216,wherein the mass spectrometry analysis is selected from the groupconsisting of atmospheric pressure ionization and laser desorptionionization.
 218. A method of liquid chromatographic analysis comprising:providing the system according to claim 177, wherein the device toprovide at least one sample in solution or fluid or combination thereofto at least one entrance orifice of said electrospray device is a liquidchromatography device; passing a fluid through the liquid chromatographydevice so that the fluid is subjected to liquid chromatographicseparation; and passing a fluid into the entrance orifice, through thechannel, and through the exit orifice of at least one spray unit underconditions effective to produce an electrospray.
 219. A method of massspectrometric analysis comprising: providing the system of claim 181,wherein the device to receive multiple sprays of fluid from saidelectrospray device is a mass spectrometer and the device to provide atleast one sample in solution or fluid or combination thereof to at leastone entrance orifice of said electrospray device is a liquidchromatography device; passing a fluid through the liquid chromatographydevice so that the fluid is subjected to liquid chromatographicseparation; passing a fluid into the entrance orifice, through thechannel, and through the exit orifice of at least one spray unit underconditions effective to produce an electrospray; and passing theelectrospray into the mass spectrometer, whereby the fluid is subjectedto a mass spectrometry analysis.
 220. A method of generating anelectrospray comprising: providing an electrospray system according toclaim 67; passing a fluid into the entrance orifice, through thechannel, and through the exit orifice of at least one spray unit;generating an electric field surrounding the exit orifice such thatfluid discharged from the exit orifice of said at least one spray unitforms an electrospray.
 221. The method according to claim 220, furthercomprising: detecting components of the electrospray by spectroscopicdetection.
 222. The method according to claim 221, wherein thespectroscopic detection is selected from the group consisting of UVabsorbance, laser induced fluorescence, and evaporative lightscattering.
 223. The method according to claim 220, wherein the fluid isdischarged at a flow rate of up to about 2 μL/minute.
 224. The methodaccording to claim 220, wherein the fluid is discharged at a flow rateof greater than about 2 μL/minute.
 225. The method according to claim220, wherein the fluid is discharged at a flow rate of from about 2μL/minute to about 1 mL/minute.
 226. The method according to claim 220,wherein the fluid is discharged at a flow rate of from about 100nL/minute to about 500 nL/minute.
 227. A method of mass spectrometricanalysis comprising: providing the system according to claim 170,wherein the device to receive multiple sprays of fluid from saidelectrospray device is a mass spectrometer; passing a fluid into theentrance orifice, through the channel, and through the exit orifice ofat least one spray unit under conditions effective to produce anelectrospray; and passing the electrospray into the mass spectrometer,whereby the fluid is subjected to a mass spectrometry analysis.
 228. Themethod according to claim 227, wherein the mass spectrometry analysis isselected from the group consisting of atmospheric pressure ionizationand laser desorption ionization.
 229. A method of liquid chromatographicanalysis comprising: providing the system according to claim 191,wherein the device to provide at least one sample in solution or fluidor combination thereof to at least one entrance orifice of saidelectrospray system is a liquid chromatography device; passing a fluidthrough the liquid chromatography device so that the fluid is subjectedto liquid chromatographic separation; and passing a fluid into theentrance orifice, through the channel, and through the exit orifice ofat least one spray unit under conditions effective to produce anelectrospray.
 230. A method of mass spectrometric analysis comprising:providing the system of claim 195, wherein the device to receivemultiple sprays of fluid from said electrospray system is a massspectrometer and the device to provide at least one sample in solutionor fluid or combination thereof to at least one entrance orifice of saidelectrospray system is a liquid chromatography device; passing a fluidthrough the liquid chromatography device so that the fluid is subjectedto liquid chromatographic separation; passing a fluid into the entranceorifice, through the channel, and through the exit orifice of at leastone spray unit under conditions effective to produce an electrospray;and passing the electrospray into the mass spectrometer, whereby thefluid is subjected to a mass spectrometry analysis.
 231. A method ofgenerating multiple sprays from a single fluid stream of an electrospraydevice comprising: providing an electrospray device for spraying a fluidcomprising: a substrate having a) an injection surface; b) an ejectionsurface opposing the injection surface, wherein the substrate is anintegral monolith having a plurality of spray units wherein entranceorifices of each spray unit are in fluid communication with one another,each spray unit comprising: an entrance orifice on the injectionsurface, an exit orifice on the ejection surface, a channel extendingbetween the entrance orifice and the exit orifice, and a recesssurrounding the exit orifice positioned between the injection surfaceand the ejection surface; and c) an electric field generating sourcepositioned to define an electric field surrounding each exit orifice,wherein each spray unit generates at least one plume of the fluidcapable of overlapping with that emanating from other spray units ofsaid electrospray device; depositing on the injection surface analytefrom a fluid sample; eluting the analyte deposited on the injectionsurface with an eluting fluid; passing the eluting fluid containinganalyte into the entrance orifice, through the channel, and through theexit orifice of each spray unit; generating an electric fieldsurrounding the exit orifice such that fluid discharged from the exitorifice of each of the spray units forms an electrospray.
 232. Themethod according to claim 231, wherein said depositing on the injectionsurface comprises: contacting the fluid sample with the injectionsurface and evaporating the fluid sample under conditions effective todeposit the analyte on the injection surface.
 233. The method accordingto claim 231, wherein the substrate for said electrospray device has aplurality of spray units for spraying the fluid.
 234. The methodaccording to claim 231, wherein the fluid is discharged at a flow rateof up to about 2 μL/minute.
 235. The method according to claim 231,wherein the fluid is discharged at a flow rate of greater than about 2μL/minute.
 236. The method according to claim 231, wherein the fluid isdischarged at a flow rate of from about 2 μL/minute to about 1mL/minute.
 237. The method according to claim 231, wherein the fluid isdischarged at a flow rate of from about 100 nL/minute to about 500nL/minute.
 238. A method of mass spectrometric analysis comprising:providing a mass spectrometer and passing the electrospray produced bythe method according to claim 231 into the mass spectrometer, wherebythe fluid is subjected to a mass spectrometry analysis.
 239. The methodaccording to claim 238, wherein the mass spectrometry analysis isselected from the group consisting of atmospheric pressure ionizationand laser desorption ionization.
 240. A method of producing anelectrospray device comprising: providing a substrate having opposedfirst and second surfaces, the first side coated with a photoresist overan etch-resistant material; exposing the photoresist on the firstsurface to an image to form a pattern in the form of at least one ringon the first surface; removing the exposed photoresist on the firstsurface which is outside and inside the at least one ring leaving theunexposed photoresist; removing the etch-resistant material from thefirst surface of the substrate where the exposed photoresist was removedto form holes in the etch-resistant material; optionally, removing allphotoresist remaining on the first surface; coating the first surfacewith a second coating of photoresist; exposing the second coating ofphotoresist within the at least one ring to an image; removing theexposed second coating of photoresist from within the at least one ringto form at least one hole; removing material from the substratecoincident with the at least one hole in the second layer of photoresiston the first surface to form at least one passage extending through thesecond layer of photoresist on the first surface and into substrate;optionally removing all photoresist from the first surface; applying anetch-resistant layer to all exposed surfaces on the first surface sideof the substrate; removing the etch-resistant layer from the firstsurface that is around the at least one ring; removing material from thesubstrate exposed by the removed etch-resistant layer around the atleast one ring to define at least one nozzle on the first surface;providing a photoresist over an etch-resistant material on the secondsurface; exposing the photoresist on the second surface to an image toform a pattern circumscribing extensions of the at least one hole formedin the etch-resistant material of the first surface; removing theexposed photoresist on the second surface; removing the etch-resistantmaterial on the second surface coincident with where the photoresist wasremoved; removing material from the substrate coincident with where theetch-resistant material on the second surface was removed to form areservoir extending into the substrate to the extent needed to join thereservoir and the at least one passage; and applying an etch-resistantmaterial to all surfaces of the substrate to form the electrospraydevice.
 241. The method according to claim 240, wherein the substrate ismade from silicon and the etch-resistant material is silicon dioxide.242. The method according to claim 240 further comprising: applying asilicon nitride layer over all surfaces after said applying anetch-resistant material to all exposed surfaces of the substrate. 243.The method according to claim 242 further comprising: applying aconductive material to a desired area of the substrate.
 244. A method ofproducing an electrospray device comprising: providing a substratehaving opposed first and second surfaces, the first side coated with aphotoresist over an etch-resistant material; exposing the photoresist onthe first surface to an image to form a pattern in the form of at leastone ring on the first surface; removing the exposed photoresist on thefirst surface which is outside and inside the at least one ring leavingthe unexposed photoresist; removing the etch-resistant material from thefirst surface of the substrate where the exposed photoresist was removedto form holes in the etch-resistant material; optionally, removing allphotoresist remaining on the first surface; providing a photoresist overan etch-resistant material on the second surface; exposing thephotoresist on the second surface to an image to form a patterncircumscribing extensions of the at least one ring formed in theetch-resistant material of the first surface; removing the exposedphotoresist on the second surface; removing the etch-resistant materialon the second surface coincident with where the photoresist was removed;removing material from the substrate coincident with where theetch-resistant material on the second surface was removed to form areservoir extending into the substrate; and optionally removing theremaining photoresist on the second surface; coating the second surfacewith an etch-resistant material; coating the first surface with a secondcoating of photoresist; exposing the second coating of photoresistwithin the at least one ring to an image; removing the exposed secondcoating of photoresist from within the at least one ring to form atleast one hole; removing material from the substrate coincident with theat least one hole in the second layer of photoresist on the firstsurface to form at least one passage extending through the second layerof photoresist on the first surface and into substrate to the extentneeded to reach the etch-resistant material coating the reservoir;removing at least the photoresist around the at least one ring from thefirst surface; removing material from the substrate exposed by theremoved etch-resistant layer around the at least one ring to define atleast one nozzle on the first surface; removing from the substrate atleast the etch-resistant material coating the reservoir; and applying anetch resistant material to coat all exposed surfaces of the substrate toform the electrospray device.
 245. The method according to claim 244,wherein the substrate is made from silicon and the etch-resistantmaterial is silicon dioxide.
 246. The method according to claim 244further comprising: applying a silicon nitride layer over all surfacesafter said applying an etch-resistant material to all exposed surfacesof the substrate.
 247. The method according to claim 246 furthercomprising: applying a conductive material to a desired area of thesubstrate.
 248. A method for producing larger, minimally-chargeddroplets from a device, comprising: providing the electrospray device ofclaim 2; passing a fluid into at least one entrance orifice, through thechannel, and through the exit orifice of at least one spray unit of saidelectrospray device; and generating an electric field surrounding theexit orifice to a value less than that required to generate anelectrospray of said fluid.
 249. The method according to claim 248,wherein the fluid to substrate potential voltage ratio is less thanabout
 2. 250. A method for producing larger, minimally-charged dropletsfrom a device, comprising: providing the electrospray system of claim67; passing a fluid into at least one entrance orifice, through thechannel, and through the exit orifice of at least one spray unit of atleast one electrospray device; and generating an electric fieldsurrounding the exit orifice to a value less than that required togenerate an electrospray of said fluid.
 251. The method according toclaim 250, wherein the fluid to substrate potential voltage ratio isless than about 2.